Inhibitors and their uses

ABSTRACT

The present invention relates to inhibitors of PPP1 R15A and PPP1 R15B and their use in therapy, particularly in the treatment of a disease state alleviated by the inhibition of PPP1 R15A and PPP1 R15B, for example a disorder associated with accumulation of misfolded proteins or proteostatsis disorder. Compounds of the invention include compounds having the formula IA or a pharmaceutically acceptable salt thereof, wherein R 1a , R 3a , R 5a , X a  and Y a  are as defined herein.

The present invention relates to inhibitors of PPP1R15A and PPP1R15B andtheir use in therapy.

BACKGROUND TO THE INVENTION

The reversible phosphorylation of proteins controls virtually allaspects of cell and organismal function, allowing cells to adapt tosudden changes through the antagonistic action of kinases andphosphatases. Consequently, targeting phosphorylation offers a broadrange of therapeutic opportunities and kinases have arisen as the mostprevalent drug targets in today's pharmaceutical research with more than3000 approved and experimental drugs. However, while targetingphosphatases should in principle be as attractive as kinases, thetherapeutic potential of phosphatases has been overlooked. The majorityof protein phosphorylation occurs on serine and threonine and selectiveserine/threonine dephosphorylation is achieved by hundreds of differentdimeric or trimeric holoenzymes assembled from one of only a fewcatalytic subunits combined with one amongst hundreds of diverseregulatory subunits (Heroes et al., FEBS Journal, 280, 584-595, 2012).Thus, inhibition of the catalytic component of the holoenzyme such asPP1c results in inhibition of hundreds of phosphatases and is toxic.Since selectivity is an important property for drug development, thepromiscuity of catalytic phosphatases has led them to acquire thereputation of being undruggable.

Phosphorylation of the a subunit of elF2α is the first line of defenseagainst a variety of stresses and is thereby a central component of twopartly overlapping signaling pathways: the Unfolded Protein Response(UPR) and the Integrated Stress Response (ISR). To reverse elF2αphosphorylation, mammalian cells have two elF2α phosphatases. The elF2αphosphatases are dimeric holoenzymes that share a catalytic subunit PP1cwith about 200 other phosphatases, and are bound to one of two relatedregulatory subunits: PPP1R15A (Novoa et al., The Journal of CellBiology, 153, 1345-1355, 2001), a stress inducible protein or PPP1R15B,which is constitutively expressed (Jousse et al., The Journal of CellBiology, 163, 767-775, 2003).

Recently, the feasibility of inhibiting selectively a serine/threoninephosphatase has been demonstrated. Guanabenz (Tsaytler et al., Science,332, 91-94, 2011; Tsaytler and Bertolotti, FEBS Journal, 280, 766-770,2012) and its derivatives, some of which are disclosed in WO2014108520(Medical Research Council), were found to selectively inhibitPPP1R15A/GADD34, a stress-induced regulatory subunit of theserine/threonine protein phosphatase 1, and was proposed as a treatmentfor diseases associated with protein misfolding stress.

PPP1R15A inhibition selectively inhibits the stress-induced elF2αphosphatase composed of PPP1R15A and PP1, while sparing the highlyrelated and constitutive phosphatase PPP1R15B-PP1. PPP1R15A inhibitionprolongs elF2α phosphorylation in stressed cells and this results inprolonging translation attenuation in stressed cells. As a consequence,chaperone availability is increased in stressed cells because thechaperones that are normally engaged in assisting the folding of newlysynthetized proteins become available when translation is decreased.This favors protein folding and rescues cells from protein proteostasisdefects. Thus, in principle, PPP1R15A inhibitors could treat mammaliandiseases involving protein misfolding stress. Inhibition of PPP1R15A inmammals has an attractive therapeutic potential because inhibition ofPPP1R15A is predicted to be safe as PPP1R15A/GADD34 knock-out mice arelargely indistinguishable from wild-type mice (Marciniak et al., Genes &Development, 18, 3066-3077, 2004). However, the number of therapeuticindications that can be treated with PPP1R15A inhibitors is predicted tobe restricted to diseases where PPP1R15A is expressed and where PPP1R15Ais in the disease mode of action. Thus, inhibition of PPP1R15A may bepowerful and safe but will be restricted to diseases involving PPP1R15A.

Regardless of the limitations associated with PPP1R15A inhibition, theapproach of restoring proteostasis by fine tuning translation toincrease chaperone availability is in theory powerful, straightforwardand applicable to correct a broad range of diseases involving misfoldedproteins. As noted above, the use of PPP1R15A inhibitors will berestricted to diseases where PPP1R15A is expressed and where PPP1R15A isin the disease mode of action. This represents a serious limitation.Thus alternative approaches, of broad therapeutic potential, are neededand the present invention seeks to provide these.

SUMMARY OF THE INVENTION

Compounds which inhibit PPP1R15A and PPP1R15B can advantageously be usedto treat a wider range of diseases than compounds which selectivelyinhibit PPP1R15A.

In a first aspect, the present invention provides compounds of formulaIA:

or a pharmaceutically acceptable salt thereof, wherein:

X^(a) is N or CR^(2a);

Y^(a) is N or CR^(4a);

R^(1a) is H, F, Cl or Br;

R^(2a), R^(3a), R^(4a), R^(5a) each independently represent H, F or Cl;

with the proviso that:

when X^(a) and Y^(a) represent CR^(2a) and CR^(4a) respectively andR^(2a) and R^(4a) are both H:

-   -   R^(1a) is not F, Cl or Br when R^(3a) and R^(5a) both represent        H;    -   R^(5a) is not F or Cl when R^(1a) is Cl and R^(2a) is H;    -   R^(3a) is not F when R^(1a) is Cl and R^(5a) is H;    -   R^(3a) is not Cl when R^(1a) and R^(5a) are both H or when        R^(1a) is Cl and R^(5a) is H;    -   R^(1a), R^(3a) and R^(5a) are not all H;    -   R^(1a) is not Cl when R^(3a) is H and R^(5a) is F;

when X^(a) represents CH and Y^(a) represents CR^(4a) wherein R^(4a) isCl:

-   -   R^(1a) and R^(5a) are not both Cl;    -   R^(3a) is not Cl when R^(1a) and R^(5a) are Cl;

when X^(a) represents CR^(2a) and Y^(a) represents CR^(4a) and R^(2a)and R^(4a) are both Cl, R^(1a), R^(3a) and R^(5a) are not all H.

In one embodiment, X^(a) represents CR^(2a) and Y^(a) representsCR^(4a), wherein R^(2a) and R^(4a) each independently represent H, F orCl.

In one embodiment, three of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a)represent Cl or F and two of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a)represent H, optionally wherein at least one of R^(1a), R^(2a), R^(3a),R^(4a) and R^(5a) is F.

In one embodiment, three of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a)represent Cl and two of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a)represent H.

In one embodiment, R^(3a) is Cl or F and R^(1a), R^(2a), R^(4a) andR^(5a) are independently selected from H, F and Cl, wherein two ofR^(1a), R^(2a), R^(4a) and R^(5a) are selected from F and Cl and two ofR^(1a), R^(2a), R^(4a) and R^(5a) is H.

In one embodiment, R^(5a) is H.

In one embodiment, the compound of formula IA is in the E-isomer form.

A second aspect of the invention relates to compounds of formula IB:

or a pharmaceutically acceptable salt thereof, wherein:

X^(b) is N or CR^(2b);

Y^(b) is N or CR^(4b);

R^(1b) is H, F, Cl, or Br;

R^(2b)R^(3b), R^(4b), R^(5b) each independently represent H, F, or Cl;

with the proviso that:

when X^(b) and Y^(b) both represent N, R^(1b) and R^(3b) are not bothCl;

R^(1b) is not Br when X^(b) is CR^(2b) and R^(2b) is Cl;

when R^(3b) and R^(4b) are both H, R^(1b) and R^(2b) are not both Cl;

when X^(b) is CR^(2b) and R^(2b) and R^(3b) are both H, R^(1b) is not Clwhen Y^(b) is CR^(4b) and R^(4b) is F;

when X^(b) is CR^(2b) and Y^(b) is CR4b, R^(1b), R^(2b), R^(3b), R^(4b)and R^(5b) are not all H;

when X^(b) is CR^(2b) and Y^(b) is CR^(4b) and R^(2b), R^(3b), R^(4b)and R^(5b) are H, R^(1b) is not Cl;

when X^(b) is CR^(2b) and Y^(b) is CR^(4b) and R^(2b), R^(3b) and R^(4b)are H, R^(1b) is not Cl when R^(5b) is F;

for use in the treatment of a disease state alleviated by the inhibitionof PPP1R15A and PPP1R15B.

In one embodiment, X^(b) represents CR^(2b) and Y^(b) representsCR^(4b), wherein R^(2b) and R^(4b) each independently represent H, F orCl.

In one embodiment, three of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b)represent Cl and two of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b)represent H.

In one embodiment, three of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b)represent Cl or F and two of R^(1b), R^(2b) R^(3b), R^(4b) and R^(5b)represent H, optionally wherein at least one of R^(1b), R^(2b) R^(3b)R^(4b) and R^(5b) is F.

In one embodiment, R^(1b) is Cl or F and R^(1b), R^(2b), R^(4b) andR^(5b) are independently selected from H, F and Cl, wherein three ofR^(1b), R^(2b), R^(4b) and R^(5b) are selected from F and Cl and one ofR^(1b), R^(2b), R^(4b) and R^(5b) is H.

In one embodiment, R^(5a) is H.

In one embodiment, the compound of formula IB is in the E-isomer form.

A further aspect of the invention relates to compounds of formula IA foruse in therapy.

A further aspect of the invention relates to compounds of formula IA orformula IB for use in the treatment of a disease state alleviated by theinhibition of PPP1R15A and PPP1R15B.

A further aspect of the invention relates to use of a compound offormula IA or formula IB in the preparation of a medicament for treatinga disease state alleviated by the inhibition of PPP1R15A and PPP1R15B.

A further aspect of the invention relates to methods of treating adisease state alleviated by the inhibition of PPP1R15A and PPP1R15B in asubject in need thereof, said method comprising administering atherapeutically effective amount of a compound of formula IA or IB.

In one embodiment, the disease state alleviated by the inhibition ofPPP1R15A and PPP1R15B is a disorder associated with accumulation ofmisfolded proteins or proteostatsis disorder.

In a further embodiment, the disease is Huntington's disease,Parkinson's disease, a tauopathy, a protein trafficking disease or amyelin disorder.

In another embodiment, the disease is any polyglutamine disorder.

In a further embodiment, the disease is Distal hereditary motorneuropathy with mutations in the chaperone HSJ1.

A further aspect of the invention relates to pharmaceutical compositionscomprising a compound of formula IA or formula IB as described above,admixed with a suitable pharmaceutically acceptable diluent, excipientor carrier.

BRIEF DESCRIPTION OF THE FIGURES

Certain embodiments of the present invention will now be described, byway of example only, with reference to the drawings in which:

FIG. 1 shows the selective binding of a R15B inhibitor of Example 1 toR15B-PP1 over R15A-PP1.

FIG. 2 shows that a selective R15B inhibitor of Example 1 induces atransient phosphorylation of elF2α in cells in the absence of stress andinduces expression of R15A in cells.

FIG. 3 shows that a selective R15B inhibitor of Example 1 protects cellsfrom stress.

FIG. 4 shows the effects of a selective R15B inhibitor on elF2αphosphorylation following stress. The compound of Example 1 prolongselF2⊐ phosphorylation following stress at times where R15A is not yetexpressed.

FIG. 5 shows the tissue distribution of a compound of Example 1 whichexhibits extensive tissue distribution.

FIG. 6 shows that the treatment of mice with a compound of Example 1 (10mg/kg) is not toxic.

FIG. 7 shows that the treatment of mice with a compound of Example 1 (10mg/kg) does not cause the side effects of Guanabenz

FIG. 8 shows the induction of R15A in a mammal following treatment witha compound of Example 1.

FIG. 9 shows the effectiveness of a R15B inhibitor of Example 1 inpreventing disease in a mammal. The example used in FIG. 8 isHuntington's disease using the mouse model HD82GIn (Schilling et al.,Hum. Mol. Genet., 8, 387-407, 1999). WT: wild-type mice. Tg: HD82GIn.

FIG. 10 shows myelin internodes in red (rod shaped) from cultured dorsalroot ganglia (DRG) and nuclei in blue (spherical ‘blob’ shaped) in ofthe indicated genotype treated with vehicle or compound of Example 1.The myelin internodes in the PMP22-mutant mice are shorter. Treatmentwith a compound of Example 1 increased the length of myelin internodesin mutant DRG revealing that it improved myelination.

FIG. 11 shows blood glucose levels in obese mice db/db animals (n=5 percondition) following treatment with compound of Example 1.

FIG. 12 shows protein synthesis rates for a selective R15A inhibitor(1^(st) column-guanabenz (GBZ)), a selective R15B inhibitor (2^(nd)column-compound 16 (TST3)), a combination of GBZ and TST3 (3^(1d)column), and an R15A/B inhibitor (4^(th) column-compound 10). The figureshows that a selective R15A inhibitor doesn't inhibit protein synthesisin unstressed cell. A selective R15B inhibitor transiently inhibitsprotein synthesis in unstressed cells. Combining an R15A and an R15Binhibitor results in a persistent inhibition of protein synthesis.Likewise, an R15A/B inhibitor persistently inhibits protein synthesis. Yaxis shows the relative rates of protein synthesis. X axis shows timefollowing addition of the compounds (10 μM), in hours (h).

FIG. 13 shows an immunoblot showing that an R15A/B inhibitor (compound10 at 20 μM) induces expression of ATF4, confirming the compound ison-target effect. Time after the addition of the compound to cells inculture is shown underneath the ATF4 immunoblot (0, 2, 5, 7.5 h).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides compounds of formulaIA:

or a pharmaceutically acceptable salt thereof, wherein:

X^(a) is N or CR^(2a);

Y^(a) is N or CR^(4a);

R^(1a) is H, F, Cl or Br;

R^(2a), R^(3a), R^(4a), R^(5a) each independently represent H, F or Cl;

with the proviso that:

when X^(a) and Y^(a) represent CR^(2a) and CR^(4a) respectively andR^(2a) and R^(4a) are both H:

-   -   R^(1a) is not F, Cl or Br when R^(3a) and R^(5a) both represent        H;    -   R^(5a) is not F or Cl when R^(1a) is Cl and R^(2a) is H;    -   R^(3a) is not F when R^(1a) is Cl and R^(5a) is H;    -   R^(3a) is not Cl when R^(1a) and R^(5a) are both H or when        R^(1a) is Cl and R^(5a) is H;    -   R^(1a), R^(3a) and R^(5a) are not all H;    -   R^(1a) is not Cl when R^(3a) is H and R^(5a) is F;

when X^(a) represents CH and Y^(a) represents CR^(4a) wherein R^(4a) isCl:

-   -   R^(1a) and R^(5a) are not both Cl;    -   R^(3a) is not Cl when R^(1a) and R^(5a) are Cl;

when X^(a) represents CR^(2a) and Y^(a) represents CR^(4a) and R^(2a)and R^(4a) are both Cl, R^(1a), R^(3a) and R^(5a) are not all H.

In one embodiment, R^(1a) is F, Cl or Br.

In another embodiment, R^(3a) is F or Cl.

In another embodiment, R^(5a) is H.

In one embodiment, when X^(a) represents CR^(2a) and Y^(a) representsCR^(4a), two R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represent Cl.

In one embodiment, when X^(a) represents CR^(2a) and Y^(a) representsCR^(4a), one of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represents Cland one of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represents F.

In one embodiment, when X^(a) represents CR^(2a) and Y^(a) representsCR^(4a), one of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represents Cland one of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represents Br.

In one embodiment, R^(1a) and R^(3a) both represent Cl.

In one embodiment, when X^(a) represents CR^(2a) and Y^(a) representsCR^(4a), three of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represent Cland two of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represent H.

In one embodiment, when X^(a) represents CR^(2a) and Y^(a) representsCR^(4a), two of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represent Cl,one of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represents F, and twoof R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represent H.

In one embodiment, when X^(a) represents CR^(2a) and Y^(a) representsCR^(4a), two of R¹, R^(2a), R^(3a), R^(4a) and R^(5a) represent F, oneof R^(1a),R^(2a), R^(3a), R^(4a) and R^(5a) represents Cl and two ofR^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represent H.

In one embodiment, three of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a)represent Cl or F and two of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a)represent H, optionally wherein at least one of R^(1a), R^(2a), R^(3a),R^(4a), and R^(5a) is F.

In one embodiment, when X^(a) represents CR^(2a) and Y^(a) representsCR^(4a), R^(3a) is Cl or F, two of R^(1a), R^(2a), R^(4a) and R^(5a) areselected from F and Cl and two of R^(1a), R^(2a), R^(4a), and R^(5a) isH.

In one embodiment, when X^(a) represents N, R^(1a) and R^(3a) bothrepresent Cl.

In one embodiment,

X^(a) is CR^(2a);

Y^(a) is CR^(4a);

R^(1a) is H, F, Cl or Br;

R^(2a), R^(3a) and R^(4a) each independently represent H, F or Cl;

R^(5a) represents H;

with the proviso that:

when X^(a) and Y^(a) represent CH:

-   -   R^(1a) is not F, Cl or Br when R^(3a) and R^(5a) both represent        H;    -   R^(5a) is not F or Cl when R^(1a) is Cl and R^(2a) is H;    -   R^(3a) is not F when R^(1a) is Cl and R^(5a) is H;    -   R^(3a) is not Cl when R^(1a) and R^(5a) are both H or when        R^(1a) is Cl and R^(5a) is H;    -   R^(1a), R^(3a) and R^(5a) are not all H;    -   R^(1a) is not Cl when R^(3a) is H and R^(5a) is F;

when X^(a) represents CH and Y^(a) represents CR^(4a) wherein R^(4a) isCl:

-   -   R^(1a) and R^(5a) are not both Cl;    -   R^(3a) is not Cl when R^(1a) and R^(5a) are Cl;

when X^(a) represents CR^(2a) and Y^(a) represents CR^(4a) and R^(2a)and R^(4a) are both Cl, R^(1a), R^(3a) and R^(5a) are not all H.

In one embodiment, the compound of formula IA is in the E-isomer form.

In a second aspect, the present invention provides compounds of formulaIB for use in the treatment of a disease state alleviated by theinhibition of PPP1R15A and PPP1R15B:

or a pharmaceutically acceptable salt thereof, wherein:

X^(b) is N or CR^(2b);

Y^(b) is N or CR^(4b);

R^(1b) is H, F, Cl or Br;

R^(2b), R^(3b), R^(4b), R^(5b) each independently represent H, F or Cl;

with the proviso that:

when X^(b) and Y^(b) both represent N, R^(1a) and R^(3b) are not bothCl;

R^(1b) is not Br when X^(b) is CR^(2b) and R^(2b) is Cl;

when R^(3b) and R^(4b) are both H, R^(1b) and R^(2b) are not both Cl;

when X^(b) is CR^(2b) and R^(2b) and R^(3b) are both H, R^(1b) is not Clwhen Y^(b) is CR^(4b) and R^(4b) is F;

when X^(b) is CR^(2b) and Y^(b) is CR^(4b), R^(1b), R^(2b), R^(3b),R^(4b) and R^(5b) are not all H;

when X^(b) is CR^(2b) and Y^(b) is CR^(4b) and R^(2b), R^(3b), R^(4b)and R^(5b) are H, R^(1b) is not Cl;

when X^(b) is CR^(2b) and Y^(b) is CR^(4b) and R^(2b), R^(3b) and R^(4b)are H, R^(1b) is not Cl when R^(5b) is F;

when X^(b) is CR^(2b) wherein R^(2b) is Cl and Y^(b) is CR^(4b) whereinR^(4b) is H, R^(1b) is not Cl when R^(3b), R^(4b) and R^(5b) are H, orwhen R^(3b) and R^(4b) are H and R^(5b) is Cl.

In one embodiment, X^(b) represents CR^(2b) and Y^(b) representsCR^(4b). In an alternative embodiment, X^(b) and Y^(b) both represent N.In an alternative embodiment, X^(b) represents N and Y^(b) representsCR^(4b).

The compounds of formula IB are R15A and R15B inhibitors.

In one embodiment, R^(1b) represents F, Cl or Br.

In another embodiment, R^(3b) represents F or Cl.

In another embodiment, R^(5b) is H.

In one embodiment, when X^(b) represents CR^(2b) and Y^(b) representsCR^(4b), two of R_(1b), R^(2b), R_(3b), R^(4b) and R^(5b) represent Cl.

In one embodiment, when X^(b) represents CR^(2b) and Y^(b) representsCR^(4b), one of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b) represents Cland one of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b) represents F.

In one embodiment, R^(1b) and R^(3b) both represent Cl.

In one embodiment, when X^(b) represents CR^(2b) and Y^(b) representsCR^(4b), three of R^(1b), R^(2b), R^(2b), R^(4b) and R^(5b) representCl.

In one embodiment, when X^(b) represents CR^(2b) and Y^(b) representsCR^(4b), two of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b) represent Cland one of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b) represents F.

In one embodiment, when X^(b) represents CR^(2b) and Y^(b) representsCR^(4b), two of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b) represent Fand one of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b) represents Cl.

In one embodiment, three of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a)represent Cl or F and two of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a)represent H, optionally wherein at least one of R^(1a), R^(2a), R^(3a),R^(4a) and R^(5a) is F.

In one embodiment, when X^(b) represents N, R^(1b) and R^(3b) bothrepresent Cl.

In one embodiment,

X^(b) is CR^(2b);

Y^(b) is CR^(4b);

R^(1b) is H, F, Cl or Br;

R^(2b), R^(3b) and R^(4b) each independently represent H, F or Cl;

R^(5b) is H;

with the proviso that:

when X^(b) and Y^(b) both represent N, R^(1a) and R^(3b) are not bothCl;

R^(1b) is not Br when X^(b) is CR^(2b) and R^(2b) is Cl;

when R^(3b) and R^(4b) are both H, R^(1b) and R^(2b) are not both Cl;

when X^(b) is CR^(2b) and R^(2b) and R^(3b) are both H, R^(1a) is not Clwhen Y^(b) is CR^(4b) and R^(4b) is F;

when X^(b) is CR^(2b) and Y^(b) is CR^(4b), R^(1b), R^(2b), R^(3b),R^(4b) and R^(5b) are not all H;

when X^(b) is CR^(2b) and Y^(b) is CR^(4b) and R^(2b), R^(3b), R^(4b)and R^(5b) are H, R^(1b) is not Cl;

when X^(b) is CR^(2b) and Y^(b) is CR^(4b) and R^(2b), R^(3b) and R^(4b)are H, R^(1b) is not Cl when R^(5b) is F;

when X^(b) is CR^(2b) wherein R^(2b) is Cl and Y^(b) is CR^(4b) whereinR^(4b) is H, R^(1b) is not Cl when R^(3b),

R^(4b) and R^(5b) are H, or when R^(3b) and R^(4b) are H and R^(5b) isCl.

In one embodiment, the compound of formula IA is in the E-isomer form.

The term “PPP1R15A” is used interchangeable with the term “R15A” and theterm “PPP1R15B” is used interchangeably with the term “R15B”. Aninhibitor of PPP1R15A and PPP1R15B may be referred to as “R15A/B”,“R15AB” or “AB” throughout.

Compounds described herein include:

The E isomer forms of the compounds listed above are particularlypreferred:

Novel compounds of formula IA include:

Compound 2:2-((2,4-dichloropyrimidin-5-yl)methylene)hydrazine-1-carboximidamide

Compound 3: 2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 4: 2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 5:2-(3,5-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 6: 2-(2,3,4-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 7:2-(2,4-dichloro-5-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 9: 2-(2-bromo-3-chlorobenzylidene)hydrazine-1-carboximidamide

Compound 13:2-(2,4-dichloro-3-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 14:2-(2,3-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 18:2-(4,5-dichloro-2-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 20: 2-(2-chloro-5-fluorobenzylidene)hydrazine-1-carboximidamide

Comopund 22:2-((2,6-dichloropyridin-3-yl)methylene)hydrazine-1-carboximidamide

Compound 25: 2-(2-chloro-3-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 27:2-(2-chloro-3,4-difluorobenzylidene)hydrazine-1-carboximidamide

Compound 29:2-(2-chloro-3,5-difluorobenzylidene)hydrazine-1-carboximidamide

Compound 32:2-(2-chloro-4,5-difluorobenzylidene)hydrazine-1-carboximidamide

Novel compounds of formula IA may be selected from the following Eisomer forms:

Compound 2(E):(E)-2-((2,4-dichloropyrimidin-5-yl)methylene)hydrazine-1-carboximidamide

Compound 3(E):(E)-2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 4(E):(E)-2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 5(E):(E)-2-(3,5-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 6(E):(E)-2-(2,3,4-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 7(E):(E)-2-(2,4-dichloro-5-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 8(E):(E)-2-(4-chloro-3-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 9(E):(E)-2-(2-bromo-3-chlorobenzylidene)hydrazine-1-carboximidamide

Compound 10(E):(E)-2-(2,3,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 11(E):(E)-2-(3,4-dichlorobenzylidene)hydrazine-1-carboximidamide

Compound 13(E):(E)-2-(2,4-dichloro-3-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 14(E):(E)-2-(2,3-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 15(E):(E)-2-(3-chloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 16(E):(E)-2-(2,3-dichlorobenzylidene)hydrazine-1-carboximidamide

Compound 18(E):(E)-2-(4,5-dichloro-2-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 20(E):(E)-2-(2-chloro-5-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 21(E):(E)-2-((2-chloropyridin-3-yl)methylene)hydrazine-1-carboximidamide

Compound 22(E):(E)-2-((2,6-dichloropyridin-3-yl)methylene)hydrazine-1-carboximidamide

Compound 23(E): (E)-2-(3-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 24(E): (E)-2-(3-chlorobenzylidene)hydrazine-1-carboximidamide

Compound 25(E):(E)-2-(2-chloro-3-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 26(E):(E)-2-(2,5-dichlorobenzylidene)hydrazine-1-carboximidamide

Compound 27(E):(E)-2-(2-chloro-3,4-difluorobenzylidene)hydrazine-1-carboximidamide

Compound 29(E):(E)-2-(2-chloro-3,5-difluorobenzylidene)hydrazine-1-carboximidamide

Compound 30(E):(E)-2-(3-chloro-2-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 31(E):(E)-2-(2,3,6-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 32(E):(E)-2-(2-chloro-4,5-difluorobenzylidene)hydrazine-1-carboximidamide

Comopund 34(E):(E)-2-(3,5-dichlorobenzylidene)hydrazine-1-carboximidamide

In a preferred embodiment, the compound of formula IA is selected fromcompound 3, i.e.2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide, and compound4, i.e. 2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide, inparticular the compound of formula IA is selected from(E)-2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide and(E)-2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide.

In one embodiment, the compound of formula IA is(E)-2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide.

In another embodiment, the compound of formula IA is(E)-2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide.

Compounds of formula IB may be selected from the following:

Compound 1(E):(E)-2-((4-chlorophenyl)methylene)hydrazine-1-carboximidamide

Compound 3(E):(E)-2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 4(E):(E)-2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 5(E):(E)-2-(3,5-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 6(E):(E)-2-(2,3,4-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 7(E):(E)-2-(2,4-dichloro-5-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 8(E):(E)-2-(4-chloro-3-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 10(E):(E)-2-(2,3,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Compound 11(E):(E)-2-(3,4-dichlorobenzylidene)hydrazine-1-carboximidamide

Compound 12(E):(E)-2-(2,4-dichlorobenzylidene)hydrazine-1-carboximidamide

Compound 13(E):(E)-2-(2,4-dichloro-3-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 14(E):(E)-2-(2,3-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 15(E):(E)-2-(3-chloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 17(E): (E)-2-(2-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 18(E):(E)-2-(4,5-dichloro-2-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 21(E):(E)-2-((2-chloropyridin-3-yl)methylene)hydrazine-1-carboximidamide

Compound 22(E):(E)-2-((2,6-dichloropyridin-3-yl)methylene)hydrazine-1-carboximidamide

Compound 23(E): (E)-2-(3-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 24(E): (E)-2-(3-chlorobenzylidene)hydrazine-1-carboximidamide

Compound 25(E):(E)-2-(2-chloro-3-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 26(E):(E)-2-(2,5-dichlorobenzylidene)hydrazine-1-carboximidamide

Compound 27(E):(E)-2-(2-chloro-3,4-difluorobenzylidene)hydrazine-1-carboximidamide

Compound 29(E):(E)-2-(2-chloro-3,5-difluorobenzylidene)hydrazine-1-carboximidamide

Compound 30(E):(E)-2-(3-chloro-2-fluorobenzylidene)hydrazine-1-carboximidamide

Compound 32(E):(E)-2-(2-chloro-4,5-difluorobenzylidene)hydrazine-1-carboximidamide

In a preferred embodiment, the compound of formula IB is selected fromcompound 3, i.e.(E)-2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide, andcompound 4, i.e.(E)-2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide.

In one embodiment, the compound of formula IB is(E)-2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide.

In another embodiment, the compound of formula IB is(E)-2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide.

In one embodiment, the compounds of formula IB inhibit PPP1R15A andPPP1R15B. Inhibition of PPP1R15A and PPP1R15B can be determined frombinding analysis. For example, binding can be analysed using SPR(surface plasmon resonance) to obtain K_(D) values. A PPP1R15A andPPP1R15B inhibitor may be defined as a compound where the difference inK_(D) values between R15A and R15B is no more than about 3 fold. Inparticular, the difference in K_(D) values between R15A and R15B is nomore than about 2 fold. For example, the affinity for one target overthe other is less than about 3 fold, in particular, less than about 2fold. A “selective inhibitor” may be defined as a compound where thedifference in K_(D) values between R15A and R15B is greater than 3 fold,or even more preferably 10 or 20 fold.

In cells, the compounds of formula IB inhibit protein synthesis. UnlikeGuanabenz, an R15A inhibitor which doesn't inhibit protein synthesis inuntressed cells (Tsaytler et al., Science, 332, 91-94, 2011), or an R15Binhibitor which transiently inhibits protein synthesis , the combinationof an R15A and R15B inhibitor leads to a persistent inhibition ofprotein synthesis (FIG. 12). Likewise, an inhibitor of R15A and R15B(compound 10) persistently inhibits protein synthesis (FIG. 12). ATF4 isinduced by the inhibitor of R15A and R15B, confirming that the compoundsare on-target (FIG. 13).

Described herein is the use of compounds of formula (I), orpharmaceutically acceptable salts thereof, as PPP1R15B selectiveinhibitors:

wherein

X is N or CR²;

Y is N or CR⁴;

R¹, R², R³ and R⁴ are independently selected from H, Cl or F;

with the proviso that:

when X is CR² and Y is CR⁴ the compound of formula I is at leastmono-substituted;

when R³ is Cl, R² is not Cl;

when both R¹ and R⁴ are Cl, R² is not Cl;

when the compound of formula I is di-substituted, X is CR², Y is CR⁴ andR¹ is Cl, R³ is not Cl;

when R⁴ is Cl and the compound of formula I is di-substituted, R² is notCl;

when R¹ is F, R² is not Cl;

when X or Y is N and the compound of formula I is mono-substituted, R¹is not Cl;

when X is CR², Y is CR⁴ and the compound of formula I ismono-substituted, R¹ is not F or Cl.

The PPP1R15B selective inhibitor, or a pharmaceutically acceptable saltthereof, may be selected from:

Salts and Esters

The compounds described herein can be present as salts or esters, inparticular pharmaceutically acceptable salts or esters.

Pharmaceutically acceptable salts of the compounds described hereininclude suitable acid addition or base salts thereof. A review ofsuitable pharmaceutical salts may be found in Berge et al., J Pharm Sci,66, 1-19 (1977). Salts which are not pharmaceutically or veterinarilyacceptable may still be valuable as intermediates.

Esters are formed either using organic acids or alcohols/hydroxides,depending on the functional group being esterified.

Enantiomers/Tautomers

In all aspects of the present invention previously discussed, theinvention includes, where appropriate all enantiomers, diastereoisomersand tautomers of the compounds of the invention. The person skilled inthe art will recognise compounds that possess optical properties (one ormore chiral carbon atoms) or tautomeric characteristics. Thecorresponding enantiomers and/or tautomers may be isolated/prepared bymethods known in the art. Enantiomers are characterised by the absoluteconfiguration of their chiral centres and described by the R- andS-sequencing rules of Cahn, Ingold and Prelog. Such conventions are wellknown in the art (e.g. see ‘Advanced Organic Chemistry’, 3rd edition,ed. March, J., John Wley and Sons, New York, 1985).

Stereo and Geometric Isomers

Some of the compounds of the invention may exist as stereoisomers and/orgeometric isomers—e.g. they may possess one or more asymmetric and/orgeometric centres and so may exist in two or more stereoisomeric and/orgeometric forms. The present invention contemplates the use of all theindividual stereoisomers and geometric isomers of those inhibitoragents, and mixtures thereof. The terms used in the claims encompassthese forms, provided said forms retain the appropriate functionalactivity (though not necessarily to the same degree).

The present invention also includes all suitable isotopic variations ofthe agent or a pharmaceutically acceptable salt thereof. An isotopicvariation of an agent of the present invention or a pharmaceuticallyacceptable salt thereof is defined as one in which at least one atom isreplaced by an atom having the same atomic number but an atomic massdifferent from the atomic mass usually found in nature. Examples ofisotopes that can be incorporated into the agent and pharmaceuticallyacceptable salts thereof include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorus, sulfur, fluorine and chlorine such as ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³¹ P, ³²P, ³⁵S, ¹⁸F and ¹⁸ F and ³⁶Cl, respectively.Certain isotopic variations of the agent and pharmaceutically acceptablesalts thereof, for example, those in which a radioactive isotope such as³H or ¹⁴C is incorporated, are useful in drug and/or substrate tissuedistribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C,isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with isotopes such as deuterium,i.e., ²H, may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example, increased in vivo half-life orreduced dosage requirements and hence may be preferred in somecircumstances. For example, the invention includes compounds of formulaI where any hydrogen atom has been replaced by a deuterium atom.Isotopic variations of the agent of the present invention andpharmaceutically acceptable salts thereof of this invention cangenerally be prepared by conventional procedures using appropriateisotopic variations of suitable reagents.

Prodrugs

The invention further includes the compounds of the present invention inprodrug form, i.e. covalently bonded compounds which release the activeparent drug according to any of the exemplified compounds in vivo. Suchprodrugs are generally compounds of the invention wherein one or moreappropriate groups have been modified such that the modification may bereversed upon administration to a human or mammalian subject. Reversionis usually performed by an enzyme naturally present in such subject,though it is possible for a second agent to be administered togetherwith such a prodrug in order to perform the reversion in vivo. Examplesof such modifications include ester (for example, any of those describedabove), wherein the reversion may be carried out be an esterase etc.Other such systems will be well known to those skilled in the art.

Solvates

The present invention also includes solvate forms of the compounds ofthe present invention. The terms used in the claims encompass theseforms.

Polymorphs

The invention further relates to the compounds of the present inventionin their various crystalline forms, polymorphic forms and (an)hydrousforms. It is well established within the pharmaceutical industry thatchemical compounds may be isolated in any of such forms by slightlyvarying the method of purification and or isolation form the solventsused in the synthetic preparation of such compounds.

Therapeutic Applications

The compounds described herein have potential therapeutic applicationsin treating and preventing various diseases and disorders.

One aspect of the invention relates to compounds of formula IA for usein therapy.

Another aspect of the invention relates to a method of treating asubject having a disorder associated with accumulation of misfoldedproteins or perturbation of protein homeostasis, wherein the methodcomprises administering to the subject a therapeutically effectiveamount of a compound the invention.

Another aspect of the invention relates to a method of treating asubject having a disease state alleviated by the inhibition of PPP1R15Aand PPP1R15B, wherein the method comprises administering to the subjecta therapeutically effective amount of a compound the invention.

A further aspect of the invention relates a method of preventing adisorder associated with accumulation of misfolded proteins orperturbation of protein homeostasis in a subject, wherein the methodcomprises administering to the subject a therapeutically effectiveamount of a compound of the invention.

A further aspect of the invention relates a method of preventing adisease state alleviated by the inhibition of PPP1R15A and PPP1R15B in asubject, wherein the method comprises administering to the subject atherapeutically effective amount of a compound of the invention.

Another aspect of the invention relates to compounds of formula IA andformula IB for use in the treatment or prevention of a disorderassociated with accumulation of misfolded proteins or perturbation ofprotein homeostasis.

Another aspect of the invention relates to compounds of formula IA andformula IB for use in the treatment or prevention of a disease statealleviated by the inhibition of PPP1R15A and PPP1R15B.

Yet another aspect of the invention relates to use of a compound offormula IA and IB in the manufacture of a medicament for the treatmentor prevention of a disorder associated with accumulation of misfoldedproteins or perturbation of protein homeostasis.

Yet another aspect of the invention relates to use of a compound offormula IA and IB in the manufacture of a medicament for the treatmentor prevention of a disease state alleviated by the inhibition ofPPP1R15A and PPP1R15B.

PPP1R15A and PPP1R15B related diseases are diseases that can beameliorated by inhibiting PPP1R15A and PPP1R15B. These include disordersassociated with accumulation of misfolded proteins or perturbation ofprotein homeostasis (proteostasis) such as Huntington's disease,Parkinson's disease, Alzheimer's disease, ataxias and otherpolyglutamine disorders as well as retinal degeneration, glaucoma,amyotrophic lateral sclerosis (ALS) and prion diseases; disordersassociated with aggregation of the microtubule-associated protein tauand include Alzheimer's disease, amyotrophic lateral sclerosis andparkinsonism-dementia complex, argyrophilic grain disease, chronictraumatic encephalopathy, corticobasal degeneration, diffuseneurofibrillary tangles with calcification (DNTC), Down's syndrome,familial British dementia (FBD), familial Danish dementia (FDD),frontotemporal dementia and parkinsonism linked to chromosome 17(FTDP-17), frontotemporal lobar degeneration (FTLD),Gerstmann-Sträussler-Scheinker disease, Gaudeloupean parkinsonism,myotonic dystrophy, neurodegeneration with brain iron accumulation,Niemann-Pick disease type C, non-Guamanian motor neuron disease withneurofibrillary tangles, Pick's disease, postencephalitic parkinsonism,prion protein cerebral amyloid angiopathy, progressive subcorticalgliosis, progressive supranuclear palsy, SLC9A6-related mentalretardation, subacute sclerosing panencephalitis, tangle-only dementia,and white matter tauopathy with globular glial includsions; myelindisorders, such as multiple sclerosis, Pelizaeus-Merzbacher disease,vanishing white matter disease, acute disseminated encephalomyelitis,periventricular leukomalacia, periventricular white matter injury, TabesDorsalis, Devic's disease, optic neuritis, progressive multifocalleukoencephalopathy, transverse myelitis, chronic inflammatorydemyelinating polyneuropathyl, anti-MAG peripheral neuropathy,adrenoleukodystrophy, adrenomyeloneuropathy, diffuse white matterinjury, Guillain-Barre Syndrome, central pontine myelinolysis, inheriteddemyelinating diseases such as leukodystrophy, and Charcot Marie Toothdisease; diseases caused by the misfolding or trafficking defects of anyprotein made in the endoplasmic reticulum (ER), such as cystic fibrosis,congenital hypothyroid goieter, familial neurohypophyseal diabetes,procollagen biosynthesis disorders including osteogenesis imperfect,hypercholesterolemia, alpha-1 antitrypsin deficiencies, lysomaldisorder, retinis pigmentosa (RP), and inflammatory bowel disease;metabolic diseases, such as diabetes, Wolcott-Rallison syndrome,obesity, insulin resistance, hyperlipidemia, fatty liver disease andatherosclerosis; cancer; aging; inflammation; and other disordersincluding rheumatoid arthritis, type-1 diabetes and vitiligo.

In one preferred embodiment, the compounds described herein are for usein treating disorders associated with pathological UPR or ISR and/ordefects in protein homeostasis.

As used herein, the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

The term “therapeutically effective amount” refers to that amount of thecompound being administered which will relieve to some extent one ormore of the symptoms of the disease of disorder being treated.

Herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease ordisorder, substantially ameliorating clinical symptoms of a disease ordisorder or substantially preventing the appearance of clinical symptomsof a disease or disorder.

The phrase “manufacture of a medicament” includes the above describedcompound directly as the medicament in addition to its use in ascreening programme for further active agents or in any stage of themanufacture of such a medicament.

Diseases with Potential Protein or Peptide Misfolding and/or Aggregationin their Mode of Action

Disease-causing proteins are expressed throughout life but degenerativediseases are mostly late-onset. This suggests that the differentdisease-causing proteins gradually become detrimental over time. Whileit is now well established that misfolded proteins cause distinctdegenerative diseases, why they accumulate remains largely unclear.Cells normally strive to ensure that proteins are correctly folded andindeed all cells have powerful and sophisticated protein quality controlsystems that very efficiently handle potentially harmful proteins fordecades. However, the protein quality control mechanisms seem togradually fail with age, leading to the accumulation of misfoldedproteins with the resulting catastrophic consequences for cells andorganisms. These misfolded/aggregated proteins or peptides can bepresent inside or outside the cell and can be found at any location. Inprinciple, boosting the natural cellular defences against misfoldedproteins should represent a generic approach to reduce the pathology indiverse protein misfolding diseases where misfolded/aggregation proneproteins are present in the pathology. The present invention describessuch an approach and demonstrates both its safety and efficacy in amammal.

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson'sdisease (PD), Huntington's disease (HD), ataxias and other polyglutaminedisorders, tauopathies as well as, retinal degeneration, glaucoma,amyotrophic lateral sclerosis (ALS) and prion diseases are devastatingand affect an increasing number of individuals in the ageing population.These diseases are clinically diverse but share a common mechanism. Theyare caused by the progressive dysfunction and death of specific nervecells in selective regions of the brain due to the accumulation ofspecific proteins of aberrant shape. The misfolded and aggregation proneproteins include, but are not restricted to: Aβ42, α-synuclein, TAU,TDP-43, TLS/FUS, SOD1, Huntingtin and other proteins with polyglutamineexpansion, prions and the translation product(s) of C9ORF72.

The Applicant has demonstrated that the compound of Example 1selectively inhibits PPP1R15B-PP1, correcting a protein misfoldingdisease in mice. Inhibitors of PPP1R15B described herein therefore havetherapeutic applications in the treatment of a variety of diseases wherea misfolded protein is involved and in particular with an accumulationof misfolded proteins. Inhibitors of PPP1R15A and PPP1R15B, such as thecompounds of formula IB, are also expected to have application in thetreatment of diseases where a misfolded protein is involved.

The present invention provides for the therapy of polyglutaminedisorders. Huntington's disease belongs to a broader group of disorders,“polyglutamine disorders”, characterized by expansion of CAG codonstranslated in glutamine in unrelated proteins. Huntington's disease iscaused by an expansion in the gene encoding Huntingtin; Spinal andbulbar muscular atrophy, Dentalorubral-pallidoluysian atrophy, andSpinocerebellar ataxias are caused by expansion in genes encodingAndrogen Receptor, Atrophin 1, Ataxin 1, 2, 3, α-voltage dependentcalcium channel subunit and TBP respectively. CAG expansion istranslated in polyglutamine and causes aggregation of the affectedprotein. Accordingly, prevention and/or treatment of polyglutaminedisorders such as these are within the scope of the invention.

As an example, the Applicant has shown that the compound of Example 1ameliorates Huntington's disease in a mammal. Thus, without wishing tobe bound by theory, it is believed that an inhibitor of PPP1R15B has aprotective effect against diverse diseases caused bymisfolded/aggregated proteins such as but not restricted to Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD),ataxias and other polyglutamine disorders as well as, retinaldegeneration, glaucoma, Amyotrophic Lateral Sclerosis (ALS), tauopathiesand prion diseases. Inhibitors of PPP1R15A and PPP1R15B, such as thecompounds of formula IB, are expected to have the same effect.

The diseases include any diseases where misfolding/aggregation isinvolved with the proteins known today and described above but will alsoapply to new proteins and perhaps new diseases in the future.

In a preferred embodiment, the invention provides for the therapy ofproteostasis diseases.

In another embodiment, the compounds described herein are for use intreating a disease where accumulation of misfolded proteins is involvedin the mode of action.

In a further embodiment, the disease or disorder is Alzheimer's disease(AD), Parkinson's disease (PD), Huntington's disease (HD), ataxias orother polyglutamine disorder, retinal degeneration, glaucoma,Amyotrophic Lateral Sclerosis (ALS), tauopathies or a prion disease.

In a particular embodiment, the compounds described herein are for usein the treatment of Huntington's disease.

In another particular embodiment, the compounds described herein are foruse in the treatment of Parkinson's disease.

In one embodiment, the disease or disorder is associated withaggregation of the microtubule-associated protein tau.

The Applicant has demonstrated that the compound of Example 1selectively inhibits PPP1R15B-PP1, correcting a protein misfoldingdisease in mice. PPP1R15B inhibitors can also be useful to prevent orstop the progression of diseases that are caused by the same mechanism:accumulation of misfolded proteins. Inhibitors of PPP1R15A and PPP1R15B,such as the compounds of formula IB, are also expected to have thisapplication.

Examples of such diseases include, Alzheimer's disease, amyotrophiclateral sclerosis and, parkinsonism and dementia, argyrophilic graindisease, chronic traumatic encephalopathy, corticobasal degeneration,diffuse neurofibrillary tangles with calcification (DNTC), Down'ssyndrome, familial British dementia (FBD), familial Danish dementia(FDD), frontotemporal dementia and parkinsonism linked to chromosome 17(FTDP-17) (caused by MAPT mutations), frontotemporal lobar degeneration(FTLD) (some cases caused by C9ORF72 mutations),Gerstmann-Sträussler-Scheinker disease, Gaudeloupean parkinsonism,myotonic dystrophy, neurodegeneration with brain iron accumulation,Niemann-Pick disease, type C, non-Guamanian motor neuron disease withneurofibrillary tangles, Pick's disease, postencephalitic parkinsonism,prion protein cerebral amyloid angiopathy, progressive subcorticalgliosis, progressive supranuclear palsy, SLC9A6-related mentalretardation, subacute sclerosing panencephalitis, tangle-only dementia,white matter tauopathy with globular glial inclusions.

In one embodiment, the disease is a myelin disorder.

Myelin is an abundant protein of both the central and peripheral nervoussystem. It is produced by two cell types: oligodendrocytes in thecentral nervous system and Schwann cells in the peripheral nervoussystem. Myelin forms a sheath around axons to insure the speed ofconduction of electrical impulses along an axon, and to preventelectrical current from dissipating from the axon. Myelin function isessential for the nervous system.

Myelin disorders affect more than 2.5 million people worldwide and aredefined as any disease associated with damage in myelin. Myelindisorders are manifested by diverse symptoms including but notrestricted to motor impairments, sensory impairments, cognitivedysfunction, emotional disturbances, and impaired coordination.

There are many demyelinating disorders, the most common of which ismultiple sclerosis (MS). Multiple sclerosis is an autoimmune diseaseaffecting the brain and spinal cord resulting in demyelination in thebrain. In addition to MS, other demyelinating disorders include but arenot limited to Pelizaeus-Merzbacher disease and vanishing white matterdisease, acute disseminated encephalomyelitis, periventricularleukomalacia, periventricular white matter injury, Tabes Dorsalis,Devic's disease, optic neuritis, progressive multifocalleukoencephalopathy, transverse myelitis, chronic inflammatorydemyelinating polyneuropathy, anti-MAG peripheral neuropathy,adrenoleukodystrophy, adrenomyeloneuropathy, diffuse white matterinjury, Guillain-Barre Syndrome, central pontine myelinolysis, inheriteddemyelinating diseases such as leukodystrophy, and Charcot Marie Tooth(CMT) disease.

CMT disease is a group of myelin neuropathies caused by mutations in anumber of genes. Mutations in the peripheral myelin protein PMP22 arethe most common causes of CMT. A mutation in PMP22 (Trembler-J) causesthe misfolding of PMP22 and results in a disease in mice that resemblesCMT in human due to defects in myelin in the peripheral nervous system.The Applicants have demonstrated that the compound of Example 1 canimprove myelination in explants from neuropathic mice. The Applicantshave demonstrated that improving myelination in explants fromneuropathic mice predicts efficacy in a mammal (Das et al. Science,2015). Therefore the compound of Example 1 will be useful in treatingCMT in mammals and other myelin disorders where it is known that themechanisms are similar and involve the elF2α pathway (Lin and Popko,Nat. Neurosci., 12, 379-385, 2009) and is anticipated that inhibitors ofPPP1R15A and PPP1R15B, such as the compounds of formula (I), will alsobe useful in treating CMT.

In one embodiment, the compounds described herein are for use intreating a myelin disorder.

In another embodiment, the compounds described herein are for use intreating Charcot Marie Tooth disease.

In a further embodiment, the compounds described herein are for use intreating myelin disorders of the central nervous system, for example,multiple sclerosis. It is known that the mechanisms of CMT and MS aresimilar with an exhaustion of myelin producing cells (Schwann cells inCMT and oligodendrecytes in MS) and involve pathological signalling ofthe elF2α -RRR1R15A pathway (Lin and Popko, Nat. Neurosci., 12, 379-385,2009). Since the Applicants have demonstrated that Example 1 iseffective in a myelinopathy and have also demonstrated thebioavailability of a compound of Example 1 in both the central andperipheral nervous system (FIG. 5), it is anticipated that PPP1R15Binhibitors, and inhibitors of PPP1R15A and PPP1R15B such as thecompounds of formula IB, will be useful in treating multiple sclerosis.

In one embodiment, the disease is a disease arising as a consequence ofa mutation in a protein resulting in its misfolding and mislocalisationor trafficking defects.

The Applicants have demonstrated that the compound of Example 1 canrescue defects caused by one misfolded protein, PMP22, synthetized inthe endoplasmic reticulum (ER). Due to the mechanism of action(decreasing translation to increase folding) an inhibitor of PPP1R15B,and an inhibitor of PPP1R15A and PPP1R15B, will also be useful for thetreatment of diseases due to the misfolding or trafficking defects ofany protein made in the ER, including transmembrane or secretedproteins.

Examples of such diseases include: cystic fibrosis caused by mutationsimpairing folding of the transmembrane protein (CFTR); congenitalhypothyroid goitre with thyroglobulin deficiency due to the misfoldingand/or trafficking defect of the hormone thyroglobulin; familialneurohypophyseal diabetes insipidus caused by misfolding and absence ofcirculating arginine vasopressin (this may also include certain forms ofgenetically inherited nephrogenic diabetes insipidus); procollagenbiosynthesis disorders where the disease is caused by a failure to fold,assemble and synthetize collagen, such as, but not restricted to,osteogenesis imperfect; more generally, any genetic diseases ofconnective tissues where protein misfolding/lack of synthesis ormislocalization is in the mode of action such as growth plate dysplasiaassociated with defects of proteins from extracellular matrix;hypercholesterolemia, with molecular defects caused by mutations in theLDL receptor causing lack of synthesis, altered intracellular transport,or abnormal function; Alpha-1 antitrypsin deficiencies due to themisfolding of alpha 1 antitrypsin; lysomal disorder due to misfolding ofproteins associated for lysosomal function such as Gaucher disease andNiemann-Pick disease and Anderson-Fabry disease; retinis pigmentosa(RP), which is the most common form of hereditary retinal degenerationcaused by the misfolding of rhodopsin proteins, their ER retention andthe resulting ER stress and cell death; and inflammatory bowel diseasewhich is associated with ER stress.

For the same reasons, an inhibitor of PPP1R15B or an inhibitor ofPPP1R15A and PPP1R15B, such as the compounds of formula IB, can be usedto treat the following disorders, associated with pathological UPRand/or defects in a transmembrane protein (Lin and Popko, Nat.Neurosci., 12, 379-385, 2009). These disorders include, but are notrestricted to Pelizaeus-Merzbacher disease associated with mutations inthe membrane proteolipid protein (PLP) gene, and vanishing white matter(VWM) disease as well as multiple sclerosis, a common myelin disorder.

In one embodiment, the compounds described herein are for use in thetreatment of diseases arising from a mutation in a protein resulting inthe protein's misfolding and mislocalisation or trafficking defects.

In a another embodiment, the disease arising from a mutation in aprotein resulting in the protein's misfolding and mislocalisation ortrafficking defects is selected from cystic fibrosis, congenitalhypothyroid goitre, familial neurohypophyseal diabetes insipidus,procollagen biosynthesis disorders such as osteogenesis,hypercholesterolemia, alpha-1 antitrypsin deficiencies, lysomaldisorders such as Gaucher disease, Niemann-Pick disease andAnderson-Fabry disease, retinis pigmentosa and inflammatory boweldisease.

In one embodiment, the disease is a metabolic disease.

It is known that metabolic diseases such as diabetes, obesity, insulinresistance, hyperlipidemia, fatty liver disease, and atherosclerosis areassociated with pathological ER stress and it is believed thatpharmacological modulators of the UPR may have therapeutic benefit (Caoand Kaufman, 2012, Curr Biol, vol. 22 (16)). However, as no PPP1R15Binhibitors were previously available and PPP1R15B inhibition waspredicted to be deleterious, and furthermore, since it is challenging toinhibit phosphatases, it was unclear whether PPP1R15B could be atherapeutic target in metabolic diseases.

The inventors have demonstrated that the compound of Example 1 canameliorate a metabolic disorder in a mammal (FIG. 11). Therefore,PPP1R15B inhibitors will be useful to treat metabolic disorders such as,but not restricted to diabetes, obesity, fatty liver disease, andatherosclerosis. It is also expected that inhibitors of PPP1R15A andPPP1R15B, such as the compounds of formula (I), will also be useful intreating metabolic disorders.

In one embodiment, the compounds described herein are for use in thetreatment of metabolic disorders.

In a preferred embodiment, the metabolic disorder is selected fromdiabetes, obesity, fatty liver disease, and atherosclerosis.

PPP1R15B selective inhibitors are also useful in the treatment of otherdisorders including rheumatoid arthritis, diabetes, Wolkott Rallisonsyndrome, inflammatory bowel disease and vitiligo, which involve UPR intheir mechanism of action (Cao and Kaufman, 2012, Curr Biol, vol. 22(16)). PPP1R15A and PPP1R15B inhibitors, such as the compounds offormula (I) are also expected to be useful in treatment of thesedisorders.

Cancer

In one embodiment, a compound described herein is for use in treatingcancer.

Cancer cells have high metabolic requirement and their proliferationrelies on efficient protein synthesis. Translation initiation plays acrucial role in controlling protein homeostasis, differentiation,proliferation and malignant transformation. Increasing translationinitiation contributes to cancer initiation and conversely, decreasingtranslation initiation could reduce tumor growth (Donze et al., 1995,EMBO J, 14, 3828-34; Pervin et al., 2008, Cancer Res, 68, 4862-74; Chenet al., 2011 , Nat Chem Biol, 7, 610-6). Without wishing to be bound bytheory, it is believed that inhibiting PPP1R15A and PPP1R15B couldreduce translation in tumor cells and thus reduce tumor growth.

Aging

There is abundant literature showing that reducing protein synthesisincreases life span (Tavernarakis, 2008, Trends Cell Biol, 18 (5),228-235. Therefore it is reasonable to predict that reducing proteinsynthesis by inhibiting PPP1R15A and PPP1R15B will increase life-span.

Inflammation

Salubrinal is an inducer of elF2α phosphorylation and is thought toinhibit R15A and R15B phosphatases by an unknown mechanism. Salubrinalwas found to suppress inflammation (Hamamura et al., 2015, CellularSignalling, 27 (4), 828-835). However, Salubrinal cannot be used inhuman due to toxicity issues. It is reasonable to anticipate that theR15A/B inhibitors disclosed here will be a potential therapy fordiseases involving inflammation.

A non exhaustive set of examples of diseases associated withinflammation are: arthritis, ulcerative colitis and inflammatory boweldisease, infections associated with inflammation, fibrosis,neurodegenerative diseases associated with inflammation or broadly anyhuman diseases associated with inflammation.

In particular, diseases associated with inflammation include: rheumatoidarthritis; multiple sclerosis; inflammatory bowel disease (IBD), whichis a term mainly used to describe two conditions, ulcerative colitis andCrohn's disease; diabetes; lupus nephritis; autoimmune inner ear disease(AlED); cystic fibrosis; Graves disease; myocarditis; autoimmunehepatitis; Alzheimer's disease; Parkinson's disease; scleroderma; GallBladder Disease; Hashimoto's Thyroiditis; autoimmune reactionoriginating in the gut triggered by antibodies against thyroid enzymesand proteins; Guillain-Barre autoimmune attack of the nervous systemoften triggered by autoimmune response to external stressors such asvaccinations; Polymyalgia Rheumatica; leukemia; and asthma.

Anit-Viral Agents

The compounds described herein may be used as anti-viral agents. Proteinsynthesis is required for viral replication. Having shown that ABinhibitors inhibit protein synthesis, it is reasonable to anticipatethat they will block viral replication and be useful to preventinfectious disease in human. Indeed, by way of example, it has beenpreviously shown that salubrinal, an inducer of elF2a phosphorylationblocks the replication of Herpex simplex virus (Boyce, M., et al. (2005)Science 307 (5711), 935-939). However, Salubrinal cannot be used inhuman due to toxicity issues. Therefore, the R15A/B inhibitors disclosedherein may have a therapeutic advantage over other translationinhibitors.

The Applicants have shown that a PPP1R15B inhibitor can preventHuntington's disease. PPP1R15B is constitutively expressed and istherefore a more broadly applicable disease target. It is reasonable toanticipate that AB inhibitors administered at a dose such that targetsare only inhibited by short pulses will prevent most (if not all)protein misfolding diseases.

Pharmaceutical Compositions

According to a further aspect of the invention there is provided apharmaceutical composition comprising a compound described herein foruse in therapy combined with any pharmaceutically acceptable carrier,adjuvant or vehicle.

The term “pharmaceutical composition” in the context of this inventionmeans a composition comprising an active agent and additionally one ormore pharmaceutically acceptable excipients.

Suitable pharmaceutically acceptable excipients are known to thoseskilled in the art and generally include an acceptable composition,material, carrier, diluent or vehicle suitable for administering acompound of the invention to an animal.

In one embodiment the animal is a mammal. In another embodiment themammal is human.

Pharmaceutical formulations include those suitable for oral, topical(including dermal, buccal, ocular and sublingual) rectal or parenteral(including subcutaneous, intradermal, intramuscular and intravenous),nasal, intra-ocularly and pulmonary administration e.g., by inhalation.The formulation may, where appropriate, be conveniently presented indiscrete dosage units and may be prepared by any of the methods wellknown in the art of pharmacy.

Dosages may be varied depending on the requirements of the patient, theseverity of the condition being treated and the characteristics of theactive ingredient being employed. Determination of the effective dose iswithin the remit of the skilled person, without undue burden. Suitabledosage forms for administration to mammals, including humans aretypically in the range of up to 100 mg/kg body weight, or may be 0.1mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg for example.

According to a further aspect of the invention, there is provided aprocess for the preparation of a pharmaceutical composition as describedabove, the process comprising bringing the active compound(s) intoassociation with the carrier, for example by admixture.

In general, the formulations are prepared by uniformly and intimatelybringing into association the active agent with liquid carriers orfinely divided solid carriers or both, and then if necessary shaping theproduct. The invention extends to methods for preparing a pharmaceuticalcomposition comprising bringing a compound disclosed herein inconjunction or association with a pharmaceutically acceptable carrier orvehicle. All methods include the step of bringing into association anactive compound with liquid carriers or finely divided solid carriers orboth and then, if necessary, shaping the product into the desiredformulation.

Combinations

In a particularly preferred embodiment, the one or more compounds of theinvention are administered in combination with one or more other activeagents, for example, existing drugs available on the market. In suchcases, the inhibitors of the invention may be administeredconsecutively, simultaneously or sequentially with the one or more otheractive agents.

Combining a compound which is a R15A inhibitor with a compound which isa R15B inhibitor results in the same advantageous properties as acompound which inhibitors both R15A and R15B. Therefore, one aspect ofthe present invention provides a combination of a R15A inhibitorcompound and a R15B inhibitor compound. The combination is useful in thetreatment of a disease state alleviated by the inhibition of PPP1R15Aand PPP1R15B, for example, a disorder associated with accumulation ofmisfolded proteins or proteostatsis disorder. The combination may beguanabenz, a known R15A inhibitor, and(E)-2-(2,3-dichlorobenzylidene)hydrazine-1-carboximidamide (compound16), a R15B inhibitor.

Drugs in general are more effective when used in combination. Inparticular, combination therapy is desirable in order to avoid anoverlap of major toxicities, mechanism of action and resistancemechanism(s). Furthermore, it is also desirable to administer most drugsat their maximum tolerated doses with minimum time intervals betweensuch doses. The major advantages of combining drugs are that it maypromote additive or possible synergistic effects through biochemicalinteractions and also may decrease the emergence of resistance.

Beneficial combinations may be suggested by studying the inhibitoryactivity of the test inhibitors with agents known or suspected of beingvaluable in the treatment of a particular disorder. This procedure canalso be used to determine the order of administration of the agents,i.e. before, simultaneously, or after delivery. Such scheduling may be afeature of all the active agents identified herein.

EXAMPLES Preparation of the Compounds According to the Present Invention

The compounds according to the present invention can be preparedaccording to the following procedures, which are shown in respect of theE isomeric forms. From these methods it will be known to the skilledperson how other isomeric forms, or the racemate, could be obtained.

Compound 1-(E)-2-((4-chlorophenyl)methylene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity M.W. Mole ratio 1 4-chlorobenzaldehyde 0.060 g140.57 0.00042 1.00 2 Aminoguanidine 0.047 g 110.55 0.00042 1.00hydrochloride 3 Sodium acetate 0.034 g 82.03 0.00042 1.00 4 Ethanol 1 ml

Procedure:

To a solution of 4-chlorobenzaldehyde (0.060 g, 0.00042 mol) in ethanol(1 ml) was sequentially added Aminoguanidine hydrochloride (0.047 g,0.00042 mol) and Sodium acetate (0.034 g, 0.00042 mol) at 25° C. Theresulting reaction mixture was heated at 80° C. for next 6 hours.Reaction completion was monitored on TLC using dichloromethane/methanol(8/2) as mobile phase. After completion of reaction, the reactionmixture was allowed to cool down to 25° C. and dumped in a saturatedsolution of NaHCO₃ (10 ml). The resulting precipitates were filtered offunder vacuum and washed with water (5 ml). The resulting solid materialwas triturated with diethyl ether (2×2 ml) and dried under vacuum toprovide the titled compound (0.040 g, 47.8% yield).

Compound2-(E)-2-((2,4-dichloropyrimidin-5-yl)methylene)hydrazine-1-carboximidamide

Reaction Scheme:

Experimental Details:

To a solution of Compound-14 (0.1 g, 0.56 mmol) in methanol (2 mL) wasadded amino guanidine hydrochloride (0.50 g, 0.45 mmol). The reactionmixture was stirred for 2 h at 65-70° C. The reaction showed 80%conversion on TLC. Reaction mixture was concentrated under reducedpressure to provide crude product which was stirred with ethyl acetate(30 mL) to give solid material which was filtered out and then driedunder vacuum. Obtained solid material was further purified by Prep HPLCpurification to afford the title compound as yellow solid (0.028 g,27.12%).

Compound 3-(E)-2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity M.W. Mole ratio 1 3,4,5-trichlorobenzaldehyde0.040 g 207.92 0.00019 1.00 2 Aminoguanidine 0.021 g 110.55 0.00019 1.00hydrochloride 3 Sodium acetate 0.015 g 82.03 0.00019 1.00 4 Ethanol 1 ml

Procedure:

To a solution of 3,4,5-trichlorobenzaldehyde (0.040 g, 0.00019 mol) inethanol (1 ml) was sequentially added amino guanidine hydrochloride(0.021 g, 0.00019 mol) and sodium acetate (0.015 g, 0.00019 mol) at 25°C. The resulting reaction mixture was heated at 80° C. for next 6 hours.Reaction completion was monitored on TLC using dichloromethane/methanol(8/2) as mobile phase. After completion of reaction, the reactionmixture was allowed to cool down to 25° C. and dumped in a saturatedsolution of NaHCO₃ (10 ml). The resulting precipitates were filtered offunder vacuum and washed with water (5 ml). The resulting solid materialwas triturated with diethyl ether (2×2 ml) and dried under vacuum toprovide the title compound (0.036 g, 70.88% yield). 1H-NMR (DMSO-d6): δ(ppm) 7.96 (s, 2H); 7.89 (s, 1H); 6.20 (brs, 2H); 5.69 (brs, 2H); MS(ESI+): m/z=265.1 [M+H]+

Compound 4-(E)-2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Synthesis of 1,2,4-trichloro-5-iodobenzene [Intermediate-1]

Calculation:

Mole Chemicals Quantity M.W. Mole ratio 1 2,4,5-trichloroaniline 4.00 g194.94 0.02051 1.00 2 NaNO₂ 1.41 g 69 0.02051 1.00 3 KI 3.74 g 1660.02251 1.10 4 Na₂S₂O₃•5H₂O 1.50 g 248.1 0.00610 0.30 5 Acetic acid 80ml 6 Conc. HCl 4 ml 7 D.M. Water 4.8 ml

Procedure:

A suspension of 2,4,5-trichloroaniline (4.00 g, 0.02051 mol) in aceticacid (80 ml) was heated at 35° C. till the mixture became clear. Afterthat the resulting reaction mixture was allowed to cool down to 25° C.Concentrated HCl (4 ml) was added in to the reaction mixture at 25° C.and the reaction mixture was stirred for next 30 minutes at the sametemperature. There after a solution of NaNO₂ (1.41 g, 0.02051 mol) inD.M. water (4.8 ml) was added in to the reaction mixture at 25° C. andthe resulting reaction mixture was stirred for 30 minutes at the sametemperature. The insoluble material was removed by filtration and washedwith acetic acid (3×15 ml). KI (3.74 g, 0.02251 mol) was added in to thecombined clear filtrate at 25° C. The resulting mixture was stirred at25° C. for 30 minutes, thereafter Na₂S₂O₃.5H₂O (1.50 g, 0.00610) wasadded in to the reaction mixture which was further stirred at 25° C. for30 minutes. The final insoluble solids in the reaction mixture materialwere removed by filtration and was washed with acetic acid (3×10 ml).The combined clear filtrate was evaporated to dryness to getIntermediate-1 (5 g, 79.6% yield) which was used for the next stepwithout any further processing.

Synthesis of Tert-Butyl (E)-3-(2,4,5-trichlorophenyl)acrylate[Intermediate-2]

Calculation:

Mole Chemicals Quantity M.W. Mole ratio 1 Intermediate-1 5.00 g 305.820.01634 1.00 2 Tert butyl acrylate 8.30 g 128.1 0.06539 4.00 3 Tributylamine 9.00 g 185.3 0.04904 3.00 4 Triphenylphosphine 0.42 g 262.20.00162 0.10 5 Palladium (II)Acetate 0.73 g 224.0 0.00325 0.20 6 DMF 30ml

Procedure:

To a solution of Intermediate-1 (5.00 g, 0.01634 mol) in DMF (30 ml)were sequentially added Tert butyl acrylate (8.30 g, 0.06539 mol), Tributylamine (9.00 g, 0.04904 mol), Triphenylphosphine (0.42 g, 0.00162mol) and Palladium (II) Acetate (0.73 g, 0.00325 mol) at 25° C. Theresulting reaction mixture was heated at 80° C. for next 3 hours.Reaction completion was monitored on TLC using n-hexane/ethyl acetate(9/1) as mobile phase. After completion of reaction, the reactionmixture was allowed to cool down to 25° C.The resulting reaction mixturewas evaporated to dryness. The resulting residue was suspended in toD.M. water (200 ml) and extracted by ethyl acetate (3×250 ml). Theresulting combined organic layer was washed with brine (100 ml), driedover Na₂SO₄ and concentrated under vacuum to get desired Intermediate-2(3.3 g, 65.96% yield) which was used for the next step without anyfurther processing.

Synthesis of 2,4,5-trichlorobenzaldehyde [Intermediate-3]

Calculation:

Mole Chemicals Quantity M.W. Mole ratio 1 Intermediate-2 0.25 g 3060.00081 1.00 2 Dimethyl sulfide 0.32 ml 3 DCM 30 ml 4 Methanol 20 ml

Procedure:

Intermediate-2 (0.25 g, 0.00081 mol) was taken up in a mixture ofmethanol: dichloromethane (2:3) (50 ml) and the reaction mixture wascooled to −78° C. with the help of a dry ice/acetone bath. Ozone gas waspurged in to the reaction mixture at −78° C. till the reaction becameblue. After that Oxygen gas was purged in to the reaction mixture for 10minutes at −78° C. followed by addition of Dimethyl sulfide (0.32 ml) at−78° C.The resulting reaction mixture was then allowed to warm to roomtemperature and allowed to stir for 10 minutes. Reaction completion wasmonitored on TLC using n-hexane/ethyl acetate (9/1) as mobile phase.After completion of reaction, the reaction mixture was evaporated todryness. The resulting residue was suspended in to the D.M. water (50ml) and extracted by diethyl ether (3×20 ml). The resulting combinedorganic layer was dried over Na₂SO₄ and concentrated under vacuum to getdesired Intermediate-3 (0.25 g, quantitative) which was used for thenext step without any further processing.

Synthesis of(E)-2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Calculation:

Mole Chemicals Quantity M.W. Mole ratio 1 Intermediate-3 0.040 g 207.920.00019 1.00 2 Aminoguanidine 0.021 g 110.55 0.00019 1.00 hydrochloride3 Sodium acetate 0.015 g 82.03 0.00019 1.00 4 Ethanol 1 ml

Procedure:

To a solution of Intermediate-3 (0.040 g, 0.00019 mol) in ethanol (1 ml)was sequentially added amino guanidine hydrochloride (0.021 g, 0.00019mol) and sodium acetate (0.015 g, 0.00019 mol) at 25° C. The resultingreaction mixture was heated at 80° C. for next 6 hours. Reactioncompletion was monitored on TLC using dichloromethane/methanol (8/2) asmobile phase. After completion of reaction, the reaction mixture wasallowed to cool down to 25° C. and dumped in a saturated solution ofNaHCO₃ (10 ml). The resulting precipitates were filtered off undervacuum and washed with water (5 ml). The resulting solid material wastriturated with diethyl ether (2×2 ml) and dried under vacuum to providethe title compound (0.04 g, 78.76% yield). ¹H-NMR (DMSO-d₆): δ (ppm)8.43 (s, 1H); 8.12 (s, 1H); 7.77 (s, 1H); 6.33 (brs, 2H); 5.83 (brs,2H); MS (ESI+): m/z=265.17 [M+H]⁺

Compound5-(E)-2-(3,5-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

To a solution of 3,5-chloro-4-fluorobenzaldehyde (0.1 g, 0.52 mmol) inmethanol (2 mL) was added amino guanidine hydrochloride (0.048 g, 0.43mmol). The reaction was stirred for 2 h at 65-70° C. The reaction showed80% conversion on TLC. Reaction mixture was cooled and concentratedunder reduced pressure to provide crude product which was stirred withethyl acetate (30 mL) to give solid material which was filtered anddried under vacuum. The solid material obtained was further purified byPrep HPLC purification to afford the title compound as white solid(0.056 g, 54.24%).

Compound 6-(E)-2-(2,3,4-trichlorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity Mol. Wt Mole ratio Intermediate 1 0.1 g 209.50.00047 1 Intermediate 2 0.052 g 110.5 0.00047 1 NaOAc 0.039 g 82.030.00047 1 Ethanol 1 mL — — 10 V

Procedure

To the solution of Intermediate 1 (0.1 g, 0.00047 mol) and Intermediate2 (0.052 g, 0.00047 mol) in ethanol (1 mL) was added NaOAc (0.039 g,0.00047 mol) and heated to 80° C. for 4 h. The progress of reaction wasmonitored by TLC using 20% MeOH in DCM as mobile phase. The coldsolution of NaHCO₃ (5 mL) was added in to the reaction mixture andstirred for 10 min. The precipitate were filtered, washed thoroughlywith water (2×10 mL) and dried under reduced pressure to obtain pureproduct which was subjected for analysis (0.097 g, 76.98% yield).

Compound7-(E)-2-(2,4-dichloro-5-fluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

To a solution of 2,4-Dichloro-5-fluorobenzaldehyde (0.1 g, 0.52 mmol) inmethanol (2 mL) was added amino guanidine hydrochloride (0.046 g, 0.42mmol). The reaction mixture was stirred for 2 h at 65-70° C. Reactionmixture was cooled and methanol was removed under reduced pressure toprovide crude product which was crystallized with ethyl acetate (30 mL)to give solid material. The obtained solid material was further purifiedby Prep HPLC purification to afford the title compound as off whitesolid (0.1 g, 96.86%).

Compound8-(E)-2-(4-chloro-3-fluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

To a solution of 4-chloro-3-fluorobenzaldehyde (0.1 g, 0.63 mmol) inmethanol (2 mL) was added amino guanidine hydrochloride (0.056 g, 0.51mmol). The reaction was stirred for 2 h at 65-70° C. The reaction showed90% conversion on TLC. Reaction mixture was concentrated under reducedpressure to provide crude product which was stirred with ethyl acetate(30 mL) to give solid material which was filtered and dried undervacuum. Further purification of the solid material obtained was carriedout with column chromatography using silica gel (100-200) and 8%MeOH/DCM as eluent to afford the title compound as off white solid(0.059 g, 54.48%).

Compound9-(E)-2-(2-bromo-3-chlorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

To a solution of 2-bromo-3-chlorobenzaldehyde (0.63 mmol) in methanol (2mL) was added amino guanidine hydrochloride (0.79 mmol). The reactionwas stirred for 2 h at 65-70° C. The reaction showed 90% conversion onTLC. Reaction mixture was concentrated under reduced pressure to providecrude product which was stirred with ethyl acetate (30 mL) to give solidmaterial which was filtered and dried under vacuum. Purification of thesolid material was carried out with column chromatography using silicagel (100-200) and 8.5% MeOH/DCM as eluent to afford the title compound.

Compound10-(E)-2-(2,3,5-trichlorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity M.W. Mole ratio 1 2,3,5-trichlorobenzaldehyde0.040 g 207.92 0.00019 1.00 2 Aminoguanidine 0.021 g 110.55 0.00019 1.00hydrochloride 3 Sodium acetate 0.015 g 82.03 0.00019 1.00 4 Ethanol 1 ml

Procedure:

To a solution of 2,3,5-trichlorobenzaldehyde (0.040 g, 0.00019 mol) inethanol (1 ml) was sequentially added amino guanidine hydrochloride(0.021 g, 0.00019 mol) and sodium acetate (0.015 g, 0.00019 mol) at 25°C. The resulting reaction mixture was heated at 80° C. for next 6 hours.Reaction completion was monitored on TLC using dichloromethane/methanol(8/2) as mobile phase. After completion of reaction, the reactionmixture was allowed to cool down to 25° C. and dumped in a saturatedsolution of NaHCO₃ (10 ml). The resulting precipitates were filtered offunder vacuum and washed with water (5 ml). The resulting solid materialwas triturated with diethyl ether (2×2 ml) and dried under vacuum toprovide the title compound (0.036 g, 70.88% yield).

Compound 11-(E)-2-(3,4-dichlorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity M.W. Mole ratio 1 3,4-dichlorobenzaldehyde 0.030g 173.96 0.00017 1.00 2 Aminoguanidine 0.019 g 110.55 0.00017 1.00hydrochloride 3 Sodium acetate 0.014 g 82.03 0.00017 1.00 4 Ethanol 1 ml

Procedure:

To a solution of 3,4-dichlorobenzaldehyde (0.030 g, 0.00017 mol) inethanol (1 ml) was sequentially added amino guanidine hydrochloride(0.019 g, 0.00017 mol) and sodium acetate (0.014 g, 0.00017 mol) at 25°C. The resulting reaction mixture was heated at 80° C. for next 6 hours.Reaction completion was monitored on TLC using dichloromethane/methanol(8/2) as mobile phase. After completion of reaction, the reactionmixture was allowed to cool down to 25° C. and dumped in a saturatedsolution of NaHCO₃ (10 ml). The resulting precipitates were filtered offunder vacuum and washed with water (5 ml). The resulting solid materialwas triturated with diethyl ether (2×2 ml) and dried under vacuum toprovide the titled compound (0.035 g, 88.2% yield).

Compound 12-(E)-2-(2,4-dichlorobenzylidene)hydrazine-1-carboximidamide

The following synthesis of compound 12 is described in Nguyen et al.,2014, 5 (10), 1075-1082

Synthetic Scheme:

Protocol:

3,5-dichlorobenzaldenhyde (1.0 mmol, 175 mg) and salt aminoguanidinehydrochloride (1.0 mmol, 110 mg) in EtOH (2 ml) were shaken at refluxfor 12 hours. After cooling at room temperature, the final compound wasrecovered as a precipitate after filtration. The title compound wasrecovered as a white powder (210 mg, 79%).

Compound13-(E)-2-(2,4-dichloro-3-fluorobenzylidene)hydrazine-1-carboximidamideSynthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity Mol. Wt Mole ratio Intermediate 1 0.1 g 193.030.00051 1 Intermediate 2 0.057 g 110.5 0.00051 1 NaOAc 0.042 g 82.030.00051 1 Ethanol 1 mL — — 10 V

Procedure:

To the solution of Intermediate 1 (0.1 g, 0.00051 mol) and Intermediate2 (0.057 g, 0.00051 mol) in ethanol (1 mL) was added NaOAc (0.042 g,0.00051 mol) and heated to 80° C. for 4 h. The progress of reaction wasmonitored by TLC using 20 % MeOH in DCM as mobile phase. The coldsolution of NaHCO₃ (5 mL) was added in to the reaction mixture andstirred for 10 min. The precipitate were filtered, washed thoroughlywith water (2×10 mL) and dried under reduced pressure to obtain pureproduct which was subjected for analysis (0.05 g; 38.75% yield).

Compound14-(E)-2-(2,3-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity Mol. Wt Mole ratio Intermediate 1 0.05 g 1930.00025 1 Intermediate 2 0.028 g 110.5 0.00025 1 NaOAc 0.021 g 82.030.00025 1 Ethanol 0.5 mL — — 10 V

Procedure:

To the solution of Intermediate 1 (0.05 g, 0.00025 mol) and Intermediate2 (0.028 g, 0.00025 mol) in ethanol (0.5 mL) was added NaOAc (0.021 g,0.00025 mol) and heated to 80° C. for 4 h. The progress of reaction wasmonitored by TLC using 20 % MeOH in DCM as mobile phase. The coldsolution of NaHCO₃ (2.5 mL) was added in to the reaction mixture andstirred for 10 min. The precipitate were filtered, washed thoroughlywith water (2×5 mL) and dried under reduced pressure to obtain pureproduct which was subjected for analysis (0.043 g, 67.18% yield).

Compound15-(E)-2-(3-chloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

To a solution of 3-chloro-4-fluorobenzaldehyde (0.1 g, 0.63 mmol) inmethanol (2 mL) was added amino guanidine hydrochloride (0.087 g, 0.79mmol). The reaction was stirred for 2 h at 65-70° C. The reaction showed80% conversion on TLC. Reaction mixture was concentrated under reducedpressure to provide crude product which was stirred with ethyl acetate(30 mL) to give solid material which was filtered and dried undervacuum. Further purification of the solid material obtained was carriedout with column chromatography using silica gel (100-200) and 8%MeOH/DCM as eluent to afford the title compound as off white solid(0.053 g, 39.15%).

Compound 16-(E)-2-(2,3-dichlorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Procedure:

To a solution of 2,3-dichlorobenzaldehyde (1 e.) in ethanol (300 ml) wassequentially added amino guanidine hydrochloride (1 eq.) and sodiumacetate (1 eq.) at 25° C. The resulting reaction mixture was heated at80° C. for next ˜6 hours. Reaction completion was monitored on TLC usingdichloromethane/methanol (8/2) as mobile phase. After completion ofreaction, the reaction mixture was allowed to cool down to 25° C. anddumped in a saturated solution of NaHCO₃ (700 ml). The resultingprecipitates were filtered off under vacuum and washed with water (100ml). The resulting solid material was triturated with diethyl ether(2×25 ml) and dried under vacuum to provide the title compound (85%yield), considering mono acetate salt) LC-MS: m/z=231.23 (M+H). ¹H-NMR(DMSO-d₆): δ (ppm) 11.85 (brs, 1H); 8.35 (s, 1H); 8.19-8.21 (dd, 1H);7.56-7.59 (dd, 1H); 7.32-7.36 (t, 1H); 7.04 (brs, 4H); 1.84 (s, 3H); MS(ESI+): m/z=231.23 [M+H]⁺

Compound 17-(E)-2-(2-fluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Procedure:

To a solution of 2-fluorobenzaldehyde (1 eq.) in ethanol (300 ml) wassequentially added aminoguanidine hydrochloride (1 eq.) and sodiumacetate (1 eq.) at 25° C. The resulting reaction mixture was heated at80° C. for next ˜6 hours. Reaction completion was monitored on TLC usingdichloromethane/methanol (8/2) as mobile phase. After completion ofreaction, the reaction mixture was allowed to cool down to 25° C. anddumped in the saturated solution of NaHCO₃ (700 ml). The resultingprecipitate were filtered off under vacuum and washed with water (100ml). The resulting solid material was titurated with diethylether (2×25ml) and dired under vacuum to provide the title compound.

Compound18-(E)-2-(4,5-dichloro-2-fluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Step-1:

  Int-2 T-3 Aluminum chloride (3.22 g, 24.24 mmol) was suspended incarbon tetrachloride (20 mL) at 0° C. and the suspension was graduallyheated. 1,2-dichloro-4-fluorobenzene (2.0 g, 12.12 mmol) was added dropwise over 2 h under reflux and the mixture was heated under refluxfurther for 0.5 h. After solution was allowed to cool to roomtemperature, mixture was carefully poured into iced water. The organiclayer was washed with water, 5% NaHCO₃ solution and then by water.Organic layer was dried over MgSO₄ and concentrated under reducedpressure to give crude intermediate. The crude intermediate wassuspended in 95% sulfuric acid and the suspension was stirred at 50° C.for 7 h. The solution was poured into iced water and the resulting crudecrystals were collected by filtration. The crystals were dissolved in 1NNaOH solution and the solution was washed with ethyl acetate. Theaqueous layer was neutralized with 6N HCl solution and was extractedwith ethyl acetate. The extracts was washed with brine, dried overanhydrous MgSO₄, and concentrated under reduced pressure. The crudecrystals thus obtained were collected by filtration (0.35 g).

Step-2:

  Int-3, T-3 To a solution of Int-2 of T-3 (0.35 g, 1.67 mmol) in THF(10 mL) was treated with borane methylsulfide complex (2.51 mmol) at0-5° C. The mixture was allowed to come to rt, stirred for 14 h at rtand quenched with sat. NaHCO₃ solution and methanol (2 mL). The mixturewas extracted with ethyl acetate. Organic layer was washed with 10% HCland saturated NaHCO₃ solutions, dried over MgSO₄ and concentrated togive 0.275 g of the product as a white solid.

Step-3:

To an ice-cold solution of Int-3 of T-3 (0.275 g, 1.41 mmol) in DCM wasadded Dess Martin periodinane (0.896 g, 2.11 mmol) and then stirred for30 min. Reaction mixture was quenched with sat. NaHCO₃ solution andstirring was quenched for 30 min. Organic layer was separated, washedwith water, dried over MgSO₄ and concentrated to provide Int-4 of T-3(0.17 g) Int-4, T-3

Step-4:

  T-3 To a solution of Compound-3 (0.15 g, 0.77 mmol) in methanol (3 mL)was added amino guanidine hydrochloride (0.069 g, 0.62 mmol). Thereaction was stirred for 2h at 65-70° C. The reaction showed 100%conversion on TLC. Reaction mixture was concentrated under reducedpressure to provide crude product which was stirred with ethyl acetate(30 mL) to give solid material which was filtered and dried undervacuum. The crude solid material was further purified by Prep HPLCpurification to afford the title compound as white solid (0.085 g,82.33%).

Compound20-(E)-2-(2-chloro-5-fluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity Mol. Wt Mole ratio Intermediate 1 0.1 g 158.60.00063 1 Intermediate 2 0.069 g 110.5 0.00063 1 NaOAc 0.051 g 82.030.00063 1 Ethanol 1 mL — — 10 V

Procedure:

To the solution of Intermediate 1 (0.1 g, 0.00063 mol) and Intermediate2 (0.069 g, 0.00063 mol) in ethanol (1 mL) was added NaOAc (0.051 g,0.00063 mol) and heated to 80° C. for 4 h. The progress of reaction wasmonitored by TLC using 20% in DCM as mobile phase. The cold solution ofNaHCO₃ (5 mL) was added in to the reaction mixture and stirred for 10min. The precipitate were filtered, washed thoroughly with water (2×10mL) and dried under reduced pressure to obtain pure product (0.086 g,63.07% yield).

Compound22-(E)-2-((2,6-dichloropyridin-3-yl)methylene)hydrazine-1-carboximidamide

Reaction Scheme:

Experimental Details:

To a solution of 2,6-Dichloronicotinaldehyde (0.1 g, 0.57 mmol) inmethanol (2 mL) was added amino guanidine hydrochloride (0.079 g, 0.71mmol). The reaction mixture was stirred for 2 h at 65-70° C. Thereaction showed 80% conversion on TLC. Reaction mixture was concentratedunder reduced pressure to provide crude residue which was stirred withethyl acetate (30 mL) to give solid material which was filtered anddried under vacuum. Further purification of the solid material wascarried out with column chromatography using silica gel (100-200) and 5%MeOH/DCM as eluent to afford the desired compound as yellow solid (0.040g, 30.99%).

Compound 23-(E)-2-(3-fluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

To a solution of 3-fluorobenzaldehyde (0.1 g, 0.81 mmol) in methanol (2mL) was added amino guanidine hydrochloride (0.047 g, 0.43 mmol). Thereaction mixture was stirred for 2 h at 65-70° C. The reaction showed90% conversion on TLC. Reaction mixture was concentrated under reducedpressure to provide crude residue which was stirred with ethyl acetate(30 mL) to give solid material which was filtered and dried undervacuum. Further purification of the solid material was carried withcolumn chromatography using silica gel (100-200) and 6.5% MeOH/DCM aseluent to afford the desired compound as off white solid (0.069 g,66.83%).

Compound 24-(E)-2-(3-chlorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity M.W. Mole ratio 1 3-chlorobenzaldehyde 0.050 g140.57 0.00035 1.00 2 Aminoguanidine 0.039 g 110.55 0.00035 1.00hydrochloride 3 Sodium acetate 0.028 g 82.03 0.00035 1.00 4 Ethanol 1 ml

Procedure:

To a solution of 3-chlorobenzaldehyde (0.050 g, 0.00035 mol) in ethanol(1 ml) was sequentially added aminoguanidine hydrochloride (0.039 g,0.00035 mol) and sodium acetate (0.028 g, 0.00035 mol) at 25° C. Theresulting reaction mixture was heated at 80° C. for next 6 hours.Reaction completion was monitored on TLC using dichloromethane/methanol(8/2) as mobile phase. After completion of reaction, the reactionmixture was allowed to cool down to 25° C. and dumped in a saturatedsolution of NaHCO₃ (10 ml). The resulting precipitates were filtered offunder vacuum and washed with water (5 ml). The resulting solid materialwas triturated with diethyl ether (2×2 ml) and dried under vacuum toprovide the titled compound (0.035 g, 50.19% yield).

Compound25-(E)-2-(3-chloro-2-fluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

To a solution of 2-Chloro-3-fluorobenzaldehyde (0.1 g, 0.63 mmol) inmethanol (2 mL) was added amino guanidine hydrochloride (0.087 g, 0.79mmol). The reaction was stirred for 2 h at 65-70° C. The reaction showed90% conversion on TLC. Reaction mixture was concentrated under reducedpressure to provide crude product which was stirred with ethyl acetate(30 mL) to give solid material which was filtered and dried undervacuum. Purification of the solid material was carried out with columnchromatography using silica gel (100-200) and 8.5% MeOH/DCM as eluent toafford the titled compound as off white solid (0.088 g, 68.19%).

Compound 26-(E)-2-(2,5-dichlorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity M.W. Mole ratio 1 2,5-dichlorobenzaldehyde 0.045g 173.96 0.00025 1.00 2 Aminoguanidine 0.028 g 110.55 0.00025 1.00hydrochloride 3 Sodium acetate 0.020 g 82.03 0.00025 1.00 4 Ethanol 1 ml

Procedure:

To a solution of 2,5-dichlorobenzaldehyde (0.045 g, 0.00025 mol) inethanol (1 ml) was sequentially added amino guanidine hydrochloride(0.028 g, 0.00025 mol) and sodium acetate (0.020 g, 0.00025 mol) at 25°C. The resulting reaction mixture was heated at 80° C. for next 6 hours.Reaction completion was monitored on TLC using dichloromethane/methanol(8/2) as mobile phase. After completion of reaction, the reactionmixture was allowed to cool down to 25° C. and dumped in a saturatedsolution of NaHCO₃ (10 ml). The resulting precipitates were filtered offunder vacuum and washed with water (5 ml). The resulting solid materialwas triturated with diethyl ether (2×2 ml) and dried under vacuum toprovide the title compound (0.039 g, 65.55% yield).

Compound27-(E)-2-(2-chloro-3,4-difluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity Mol. Wt Mole ratio Intermediate 1 0.04 g 176.550.00022 1 Intermediate 2 0.025 g 110.5 0.00022 1 NaOAc 0.018 g 82.030.00022 1 Ethanol 0.4 mL — — 10 V

Procedure:

To the solution of Intermediate 1 (0.04 g, 0.00022 mol) and Intermediate2 (0.025 g, 0.00022 mol) in ethanol (0.4 mL) was added NaOAc (0.018 g,0.00022 mol) and heated to 80° C. for 4 h. The progress of reaction wasmonitored by TLC using 20% MeOH in DCM as mobile phase. The coldsolution of NaHCO₃ (2.5 mL) was added in to the reaction mixture andstirred for 10 min. The precipitate were filtered, washed thoroughlywith water (2×5 mL) and dried under reduced pressure to obtain pureproduct which was subjected for analysis (0.063 g, 95.43% yield).

Compound29-(E)-2-(2-chloro-3,5-difluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity Mol. Wt Mole ratio Intermediate 1 0.06 g 176.550.00033 1 Intermediate 2 0.037 g 110.5 0.00033 1 NaOAc 0.027 g 82.030.00033 1 Ethanol 0.6 mL — — 10 V

Procedure:

To the solution of Intermediate 1 (0.06 g, 0.00033 mol) and Intermediate2 (0.037 g, 0.00033 mol) in ethanol (0.6 mL) was added NaOAc (0.027 g,0.00033 mol) and heated to 80° C. for 4 h. The progress of reaction wasmonitored by TLC using 20% MeOH in DCM as mobile phase. The coldsolution of NaHCO₃ (2.7 mL) was added in to the reaction mixture andstirred for 10 min. The precipitate were filtered, washed thoroughlywith water (2×2.7 mL) and dried under reduced pressure to obtain pureproduct which was subjected for analysis (0.04 g, 50.63% yield).

Compound30-(E)-2-(3-chloro-2-fluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

To a solution of 3-Chloro-2-fluorobenzaldehyde (0.1 g, 0.63 mmol) inmethanol (2 mL) was added amino guanidine hydrochloride (0.087 g, 0.79mmol). The reaction was stirred for 2 h at 65-70° C. The reaction showed90% conversion on TLC. Reaction mixture was concentrated under reducedpressure to provide crude product which was stirred with ethyl acetate(30 mL) to give solid material which was filtered and dried undervacuum. Purification of the solid material was carried out with columnchromatography using silica gel (100-200) and 8.5% MeOH/DCM as eluent toafford title compound.

Compound31-(E)-2-(2,3,6-trichlorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

To a solution of 2,3,6-trichlorobenzaldehyde (0.040 g, 0.00019 mol) inethanol (1 ml) was sequentially added amino guanidine hydrochloride(0.021 g, 0.00019 mol) and sodium acetate (0.015 g, 0.00019 mol) at 25°C. The resulting reaction mixture was heated at 80° C. for next 6 hours.Reaction completion was monitored on TLC using dichloromethane/methanol(8/2) as mobile phase. After completion of reaction, the reactionmixture was allowed to cool down to 25° C. and dumped in a saturatedsolution of NaHCO₃ (10 ml). The resulting precipitates were filtered offunder vacuum and washed with water (5 ml). The resulting solid materialwas triturated with diethyl ether (2×2 ml) and dried under vacuum toprovide the title compound.

Compound32-(E)-2-(2-chloro-4,5-difluorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity Mol. Wt Mole ratio Intermediate 1 0.1 g 176.550.00056 1 Intermediate 2 0.062 g 110.5 0.00056 1 NaOAc 0.046 g 82.030.00056 1 Ethanol 1 mL — — 10 V

Procedure:

To the solution of Intermediate 1 (0.1 g, 0.00056 mol) and Intermediate2 (0.062 g, 0.00056 mol) in ethanol (1 mL) was added NaOAc (0.046 g,0.00056 mol) and heated to 80° C. for 4 h. The progress of reaction wasmonitored by TLC using 20% MeOH in DCM as mobile phase. The coldsolution of NaHCO₃ (5 mL) was added in to the reaction mixture andstirred for 10 min. The precipitate were filtered, washed thoroughlywith water (2×10 mL) and dried under reduced pressure to obtain pureproduct which was subjected for analysis (0.117 g, 89.31% yield).

Compound 34-(E)-2-(3,5-dichlorobenzylidene)hydrazine-1-carboximidamide

Synthetic Scheme:

Experimental Details:

Calculation:

Mole Chemicals Quantity M.W. Mole ratio 1 3,5-dichlorobenzaldehyde 0.030g 173.96 0.00017 1.00 2 Aminoguanidine 0.019 g 110.55 0.00017 1.00hydrochloride 3 Sodium acetate 0.013 g 82.03 0.00017 1.00 4 Ethanol 1 ml

Procedure:

To a solution of 3,5-dichlorobenzaldehyde (0.030 g, 0.00017 mol) inethanol (1 ml) was sequentially added amino guanidine hydrochloride(0.019 g, 0.00017 mol) and sodium acetate (0.013 g, 0.00017 mol) at 25°C. The resulting reaction mixture was heated at 80° C. for next 6 hours.Reaction completion was monitored on TLC using dichloromethane/methanol(8/2) as mobile phase. After completion of reaction, the reactionmixture was allowed to cool down to 25° C. and dumped in a saturatedsolution of NaHCO₃ (10 ml). The resulting precipitates were filtered offunder vacuum and washed with water (5 ml). The resulting solid materialwas triturated with diethyl ether (2×2 ml) and dried under vacuum toprovide the title compound (0.038 g, 95.8% yield).

General procedure A:

To a solution of benzaldehyde (1 eq.) in ethanol (300 ml) wassequentially added a minoguanidine hydrochloride (1 eq.) and sodiumacetate (1 eq.) at 25° C. The resulting reaction mixture was heated at80° C. for next ˜6 hours. Reaction completion was monitored on TLC usingdichloromethane/methanol (8/2) as mobile phase. After completion ofreaction, the reaction mixture was allowed to cool down to 25° C. anddumped in the saturated solution of NaHCO₃ (700 ml). The resultingprecipitate were filtered off under vacuum and washed with water (100ml). The resulting solid material was titurated with diethylether (2×25ml) and dried under vacuum to provide the desired substitutedaminoguanidine derivative.

The following compounds were prepared according to general procedure A:

Example 1 (Compound 16(E)):(E)-2-(2,3-dichlorobenzylidene)hydrazine-1-carboximidamide

Prepared following general procedure A from 2,3-dichlorobenzaldehyde in85% yield (considering mono acetate salt) LC-MS: m/z=231.23 (M+H).¹H-NMR (DMSO-d₆): δ (ppm) 11.85 (brs, 1H); 8.35 (s, 1H); 8.19-8.21 (dd,1H); 7.56-7.59 (dd, 1H); 7.32-7.36 (t, 1H); 7.04 (brs, 4H); 1.84 (s,3H); MS (ESI+): m/z=231.23 [M+H]⁺

Example 2(E)-2-(2-chloro-4-fluorobenzylidene)hydrazine-1-carboximidamide

Prepared following general procedure A from2-chloro-4-fluorobenzaldehyde in 67% yield. ¹H-NMR (DMSO-d₆): δ (ppm)5.80 (brs, 2H); 5.84 (brs, 2H); 7.19-7.34 (m, 4H); 8.16 (s, 1H); MS(ESI+): m/z=215.1 [M+H]⁺

The protocols and assays of Example A were carried out in relation toPPP1R15B inhibition. However, it will be appreciated that the methodsdescribed herein are equally applicable to identifying/analysingPPP1R15A and PPP1R15B inhibitors.

Example A

Protein Expression, Purification and Analysis by Surface PlasmonResonance

Protein Expression and Purification

PPP1R15A³²⁵⁻⁶³⁶ and PPP1R15B³⁴⁰⁻⁶⁹⁸ were expressed and purified asfollows: the cDNAs encoding amino acids 325-636 of PPP1R15A and 340-698of PPP1R15B were His-tagged and cloned into pMAL-c5x. RecombinantPPP1R15A/B were expressed in BL21-Gold cells and purified by affinitychromatography on a HisTrap HP column (GE Healthcare), followed by aMBPTrap HP column (GE Healthcare). The proteins were analyzed on BOLTSDS-PAGE 4-12% Bis-Tris gels (Life Technologies) stained with SimplyBlueSafeStain (Life Technologies). cDNA encoding for human PP1γ was clonedinto the baculovirus transfer vector pDW464 to add a biotin acceptorpeptide (BAP). The vector also encodes for the E. coli biotin holoenzymesynthetase (BirA), so that BAP-tagged proteins can be biotinylated invivo in Spodoptere frugiperda (Sf9) insect cells (Duffy et al., Anal.Biochem., 262, 122-128, 1998). The Bac-to-Bac baculovirus expressionsystem (Life Technologies) was used to generate the recombinant bacmidDNA and Sf9 insect cells were used to amplify the viral stocks Cultureswere harvested by centrifuging at 1,200 g for 15 minutes, cell pelletswere resuspended in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.2%Triton, 5% Glycerol, 1 PiC tablet (Roche) per 50 ml and 0.2 mM PMSF) andfollowed by gentle sonication. The protein was first purified on a 5 mlHiTrap Q HP column (GE Healthcare) followed by a HiLoad 16/600 Superdex200 column (GE Healthcare). The positive fractions confirmed by SDS-PAGEand western blot were pooled, concentrate to ˜1 μM and stored at −80° C.

Capture of Biotin-PP1 on the SA Sensor Chip

A Biacore T200 (GE Healthcare) system was used for all experiments andbiotinylated PP1 was captured using a Sensor Chip SA (GE Healthcare,catalog no BR-1005-31). The streptavidin coated surface was activated by1 min injection with a solution of 50 mM NaOH and 1 M NaCl. Biotin-PP1was diluted in the running buffer (50 mM Tris pH 7.5, 100 mM NaCl, 0.1mM EGTA, 1 mM MnCl2, 0.05% Tween 20) and injected at approximately 300nM concentration directly to streptavidin coated surface for 100 sec orto reach immobilization level of biotin-PP1 corresponding to ˜7000 RU. Ablank immobilization was performed for one of the SA sensor chip surfaceto use as a reference.

Determining Steady-State Binding Constants of Small Molecules to elF2αHolophosphatase Complexes using the Biotin-PP1 Surface

With minor deviations, the same procedure and conditions were used inall binding experiments. Small molecules were stored as 50 mM stocksolutions in 100% DMSO. Prior to determining binding constants, serialdilutions of either 12 or 8 concentrations of the compounds wereprepared in the running buffer in a 96-well plate. Prior to eachcompound dilution series the regulatory subunit, R15A or R15B, wasdiluted to 15 μM in the running buffer and captured on the biotin-PP1surface, to form the holophosphatase complex on the sensor chip surface.Then, without regenerating the surface, the compound dilution series wasinjected onto the surface of the chip Sensorgrams were analyzed usingthe Biacore T200 evaluation software and the binding constantsdetermined based on a steady-state model. Kinetic experiments arecarried out using different concentrations of the compound and theirrespective equilibrium binding levels determined. These equilibriumresponse levels (Req) are plotted against concentration and fitted usinga global fit, which is able to determine steady-state affinityconstants, i.e. the concentration at 50% saturation is KD(Frostell-Karlsson et al., J. Med. Chem., 43, 1986-1992, 2000).

Mammalian Cell Culture

HeLa cells were cultured in Dulbecco's Modified Eagle's Media (DMEM)supplemented with penicillin, streptomycin, containing 5% and 10% fetalbovine serum (FBS), respectively. MEF cells were cultured in DMEMsupplemented with penicillin, streptomycin, glutamine, 55 μMβ-mercaptoethanol, 1X non-essential amino acids (Sigma-Adrich) and 10%FBS. Where indicated, cells were treated with 2.5 μg/ml tunicamycin, 1mM DTT (Sigma-Adrich) and or the indicated compounds at the indicatedconcentrations.

Protein Analyses by Immunoblots

For immunoblots, HeLa cells (80,000 cells/ml) were plated in 12-wellplates 24 hours before each experiment. Immediately after the indicatedtreatments, cells were lysed in 75 μl Laemmli Buffer, boiled at 95° C.for 5 minutes and sonicated. Proteins were separated and analysed asdescribed (Tsaytler et al., Science, 332, 91-94, 2011) with thefollowing antibodies: phospho-elf2α [pS52] and PPP1R15A/GADD34(10449-1-AP; ProteinTech Group, 1/1000 dilution) and ATF4 (Sc-200 SantaCruz).

Assessment of Cell Viability

Cells were plated in 24-well plates at a density of 15,000 (HeLa) or12,000 cells/ml (MEFs) 24 hours prior to treatment. ER stress waselicited by addition of fresh media containing 2.5 μg/ml tunicamycin(Sigma-Aldrich). Compounds of Example 1 were dissolved in DMSO and addedas indicated. DMSO was used as a mock treatment. Cell viability wasassessed by measuring the reduction of WST-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium]into formazan using Cell viability Counting Kit-8 (Dojindo) according tothe supplier's recommendation, 48 hours after tunicamycin treatment.

Toxicity of the compounds is assessed using Cell viability CountingKit-8 (Dojindo) or in a Incucyte (Essenbioscience) using both phasecontrast and IncuCyte™ Cytotox Green Reagent.

Animal Studies

All animal care and procedures were performed in compliance with theregulation on the use of Animals in Research (UK Animals ScientificProcedures Act of 1986) with local ethical approval.

For studying the effect of a compound of Example 1 on weight, C3H/B6mice were gavaged orally once a day with compounds of Example 1 and miceweight was recorded daily for 2 weeks.

To assess the efficacy of a compound of Example 1 in ameliorating adisease phenotype, 28-day old transgenic mice or littermate control wereorally administered daily with a compound of Example 1 (2 mg/kg) orvehicle for a duration of 4 weeks. Disease progression was evaluated byweighting the mice during the treatment and by assessing their motorperformances after 4 weeks of treatment.

To assess whether of a compound of Example 1 had the side effects ofGuanabenz, mice (n>3) were gavaged orally with 10 mg/kg of compound orGuanabenz. Their activity was monitored 30 minutes following dosing.Guanabenz treated mice did not move 30 minutes after dosing, due to thehypotensive activity of Guanabenz, in contrast to mice treated withcompound of Example 1 which were as active as untreated mice.

Eight weeks old HD-N171-82Q mice and their wild-type littermates werefirst habituated for 1 min on a static rotor and 1 min at constant speed(4 rpm). Habituation was repeated. The test session consisted of fourtrials with 15 min intervals in between. For each trial 5 mice wereplaced on an accelerating rotor (4 to 40 rpm) and the latency to fallwas recorded, with a maximum limit for individual animal set at 300 s.

For treatment of a metabolic disorder, Db/db animals (n=5 per condition)were treated once a day with 1 mg/kg of compound of Example 1 for threeweeks. Blood glucose levels were measured with a blood glucose meter(DSI) between 9 and 10 am. Data are mean+/−S.e.m.

Quantitative RT-PCR

RNA from brain was extracted in trizol (Life Technologies). RNAconcentration was measured using a NANODROP1000 spectrophotometer(Thermo Fisher Scientific), and 1 μg was reverse transcribed to cDNAusing a SuperScript reverse transcriptase (Life Technologies).Quantitative PCR with primers GAPDH (f): ACCACAGTCCATGCCATCAC, GAPDH(r): TCCACCACCCTGTTGCTGTA, PPP1R15A (f): CCTCCTGAAACTTGGGGACT; andPPP1R15A (r): GCTGTGATGTGGGATAAGCG was performed using SYBR® SelectMaster Mix (Ref 4472908, applied biosystems) on a Corbett Rotor-Geneversion 6000. Expression of each gene was normalized to the housekeepinggene GAPDH and expressed as fold change calculated using Paffl equation.

DRG Cultures

Dorsal root ganglia (DRG) dissected from wild-type or Pmp22Tr-J(PMP22mutant) (Henry et al., Neuropathol. Exp. Neurol., 42, 688-706,1983) embryo at 13.5 or development (E13.5) were cultured on collagencoated coverslips in neurobasal media supplemented with 4 g/l glucose, 2mM L-glutamine, 2% B27 supplement and 50 ng/ml neuronal growth factor(NGF) for 7 days. To differentiate Schwann cells and induce myelination,the cultured DRGs were then maintained in C-media (MEM mediasupplemented with 4 g/I glucose, 2 mM L-glutamine, 10% FCS, 50 ng/mlNGF). The C-media was replaced every other day with freshly added 50μg/ml ascorbic acid ±5 nM of compound of Example I and cultured for 14days for myelination by the Schwann cells. The cultured DRGs were thenfixed in 4% paraformaldehyde and immunostained with MBP (Rat Myelinbasic protein, 1/250 dilution, ab73498).

The reference for the mice is Pubmed ID: 631386.

Biochemical Assay

Assay 1: A selective PPP1R15B inhibitor selectively binds toPPP1R15B-PP1 (FIG. 1)

Surface Plasmon Resonance (SPR) was used to measure the binding affinityof compounds to the PPP1R15A/B-PP1 phosphatase complex. Biotin acceptorpeptide (BAP) was fused to the N-terminus of PP1γ, which enables thebiotinylation of the BAP-tagged protein in Sf9 insect cells. Afterpurification the BAP-PP1γ protein was captured on a streptavidin sensorchip (SA-chip, GE healthcare) to a response level of ˜5.000 RU. Usingcontrolled biotinylation enables orientated and uniform immobilizationon the sensor chip surface. The PP1γ was then used to capture PPP1R15A/Band form a holophosphatase complex on the surface of the streptavidinchip. This complex can then be used to test binding of compounds. 10 γMPPP1R15A/B protein concentration was used to form the holophosphatasecomplex during a 80 s injection. After PPP1R15A/B has been captured aconcentration series of a compound is injected over the surface of thechip, measuring the binding of the compound to the holophosphatase.After the concentration series (8 or 12 concentrations) is completed thesurface is regenerated with 3 M NaCl and we can capture R15A/B again toform a fresh holophosphatase complex to measure a binding of anothercompound concentration series. Analysing the level of equilibriumbinding as a function of concentration gives interaction affinities orsteady-state binding affinity (K_(D)).

FIG. 1 shows the K_(D) values for the compound of Example 1. A K_(D) of0.035 μM for PPP1R15B-PP1 and 1 μM for PPP1R15A-PP1 demonstrates thatthe compound of Example 1 selectively binds to the PP1R15B-PP1 complexbut that binding is significantly less to the PPP1R15A-PP1 complex.

Assay 2: A Selective PPP1R15B Inhibitor Induces a TransientPhosphorylation of elF2α in Cells, in the Absence of Stress (FIGS. 2Aand B)

The selectivity of a compound of Example 1 was revealed using an invitro binding assay with recombinant proteins. However, whilst a bindingassay can be used to screen for selective compounds binding to PPP1R15B,the properties of a compound are not necessarily predicted by a bindingassay. Thus, other assays are needed to assess whether the compoundinhibits PPP1R15B function or not.

Human cells were treated with a PPP1R15B inhibitor and elF2αphosphorylation was monitored over time. The inventors found that, inthe absence of stress (under conditions where cells do not expressPPP1R15A) treatment of cells with a compound of Example 1 induced elF2αphosphorylation. This was manifested between 1 and 7.5 hours afteraddition of the compound of Example 1. However, at 10 hours followingthe addition of the compound of Example 1, elF2α phosphorylationreturned to basal levels. This suggested that there was an active elF2αphosphatase at this time point. Indeed, the inventors noticed thatPPP1R15A was induced at the late time points following addition of thecompound of Example 1 (see Assay 3). The transient induction of elF2αphosphorylation demonstrates that the compound is a selective inhibitorof PPP1R15B in cells. Furthermore, this establishes that the compound ofExample 1 spares PPP1R15A. This assay can serve to identify otherselective PPP1R15B inhibitors.

Assay 3: A Selective PPP1R15B Inhibitor Induces Expression of PPP1R15Ain Cells (FIGS. 2A and B)

Cells and organisms usually have mechanisms to compensate fordeficiencies. The inventors therefore considered whether cells mightcompensate for PPP1R15B inhibition by inducing PPP1R15A. It has beenpreviously reported that PPP1R15A levels are increased in the liver ofPPP1R15B knock-out mice (Harding et al., Proc. Natl. Acad. Sci. U.S.A.,106, 1832-1837, 2009). It is unknown whether this compensatory responseis specific to the liver or if it can happen in cells or other tissues.Furthermore, prior to this study, it was unknown whether PPP1R15Ainduction can be observed upon pharmacological inhibition of PPP1R15B,as they were no selective inhibitors of PPP1R15B prior to this study.FIG. 2B demonstrates that cells treated with a compound of Example 1were found to induce PPP1R15A. This property, induction of PPP1R15A, canbe used as a method to screen for PPP1R15B inhibitors as compounds whichinduce PPP1R15A expression in cells will possess PPP1R15B inhibitionproperties. For example, an assay using a PPP1R15A gene or promoterfused to a reporter gene can be designed and developed in to a HTSscreen to identify compounds that induce PPP1R15A.

Assay 4: A Selective PPP1R15B Inhibitor Protects Cells from Stress (FIG.3)

FIG. 3 demonstrates that a selective PPP1R15B inhibitor protects cellsfrom stress. Cells where stressed with Tunicamycin (2.5 μg/ml) in thepresence of 0.2-5 μM of a compound of Example 1. Cell viability wasmeasured 3 days after treatment.

The inhibitor of Example 1 protects cells from cytotoxic stress causedby tunicamycin. Cytoprotection against ER stress can be measured by asuitable assay. In this instance, cytoprotection was measured in HeLacells in which ER stress was elicited by the addition of mediacontaining tunicamycin, a drug that blocks N-glycosylation, therebypreventing protein folding and inducing the unfolded protein response.Cell viability was then detected in the presence and absence of acompound of Example 1 after a set period of time, by measuring thereduction of WST-8 into formazan using the standard cell viability kitCell Viability Counting Kit-8 from Dojindo. Cytoprotection from ERstress was measured in terms of the percentage increase in viable cells(relative to control) after ER stress.

Assay 5: A Selective PPP1R15B Inhibitor Prolongs elF2a PhosphorylationDuring Stress-Recovery (FIG. 4)

The inventors reasoned that a PPP1R15B inhibitor should prolong elF2αphosphorylation following stress. To reveal this activity, it wascrucial to search for conditions where PPP1R15A is not expressed, toavoid confounding effects. The inventors took advantage of the fastkinetic and reversibility of stress induction by DTT (Bertolotti et al.,Nat. Cell. Biol., 2, 326-332, 2000; Jousse et al., 2003) and monitoredelF2α phosphorylation in cells following a 30 minute treatment with 1 mMDTT and a wash out. The inventors found that the decline in elF2αphosphorylation that normally occurs after the DTT-washout is delayedand this occurred before any substantial induction of PPP1R15A. Thus,careful monitoring of the kinetic of elF2α dephosphorylation in theearly phase of a stress-recovery paradigm such as the one described herecan be used to identify other PPP1R15B inhibitors.

Compound of Example 1 in Mice (FIG. 5) has a Good Tissue Distribution

Analysis of mouse tissues (plasma, brain, spinal cord, pancreas, liver)at different time following oral gavage of a compound of Example 1revealed that the PPP1R15B inhibitor has an extensive tissuedistribution and therefore demonstrates application in the treatment ofvarious diseases and disorders affecting different organs.

Treatment of Mice with a Compound of Example 1 is not Toxic to Mice(FIG. 6)

Mice were treated with Example 1 and monitored closely to detect anyclinical signs. It was found that mice treated for 2 weeks with up to 10mg/mg once a day of a compound of Example 1 were undistinguishable frommice treated with placebo and the mice gained weight normally (FIG. 6).This establishes that PPP1R15B inhibition is not toxic. This wassurprising and unanticipated as prior to this study one would havespeculated that PPP1R15B inhibitors would be so deleterious that theywould have no therapeutic potential.

Treatment of Mice with a Compound of Example 1 does not Cause the SideEffects Caused by Guanabenz (FIG. 7)

In humans, the adrenergic agonist activity of Guanabenz has side effectsincluding drowsiness and coma at high doses (A. H. Hall, Ann Intern Med102; 787-788; 1985). Due to of these side effects, Guanabenz is nolonger used in human. It is anticipated that Guanabenz derivatives havethe side effects of Guanabenz, associated with alpha-2 adrenergicactivity. While the structure-activity relationship of Guanabenz toalpha-2 adrenergic receptor is not available, the inventors surprisinglyfound here that Example 1 is devoid of the side effects of Guanabenz,while structurally very similar.

Assay 6: Induction of PPP1R15A in a Mammal Following Treatment with aCompound of Example 1 (FIG. 8)

PPP1R15A induction was assessed by qPCR on total mRNA extracted frombrains of mice treated with the indicated doses of compound of Example1.

Similar to what had been seen in cells, it was found that mice inducedPPP1R15A following a treatment with a compound of Example 1 and thatthis induction was dose-dependent. This explains why a selectivePPP1R15B inhibitor is tolerated in mice: PPP1R15A inductiondephosphorylates elF2α , ensuring that the elF2α phosphorylation whichresults from PPP1R15B inhibition by a compound of Example 1 is onlytransient. This is important because a persistent phosphorylation ofelF2α is detrimental. The induction of PPP1R15A in vivo by a PPP1R15Binhibitor is a pharmacodynamic parameter that can be used to evaluatethe efficacy and potency of PPP1R15B inhibitors in mammals inpre-clinical or clinical studies.

A Compound of Example 1 Prevents a Disease in a Mammal (FIG. 9)

Increasing folding by inhibition of PPP1R15B has the potential tobenefit a very broad range of human pathologies. To test this, theinventors looked at Huntington's disease (HD), a proteostasis diseasecaused by accumulation of a misfolded protein, mutant Huntingtin. Thereare some reports indicating that mutant Huntingtin induces the UPR(Duennwald and Lindquist, Gene & Development, 22, 3308-3319, 2008;Nishitoh et al., Genes & Development, 16, 1345-1355, 2002). However, theinventors' failure to detect PPP1R15A in models of Huntington's diseasesuggested that PPP1R15A is not a therapeutic target for HD. As HD has nocure to date, the inventors tested whether HD could be prevented byPPP1R15B inhibition. The inventors found that treatment of HD mice with2 mg/kg of a compound of Example 1 prevented the motor performancesimpairment (FIG. 9). This demonstrates that PPP1R15B is a validtherapeutic target and that therefore PPP1R15B inhibition will be usefulin the treatment and prevention of diseases.

As demonstrated here, to determine whether a disease can be prevented orameliorated by PPP1R15B inhibition, mouse models or humans can betreated with tolerable doses of inhibitor. To attest target inhibitionin vivo, markers of the PPP1R15B pathway can be monitored and used aspharmacodynamics markers. Such markers can be PPP1R15A, as shown here(FIG. 8) or any other on-pathway targets such as UPR or ISR markers(including but not restricted to elF2α phosphorylation, CHOP, ATF4). Asshown here with a compound of Example 1, a PPP1R15B inhibitor will beuseful for therapies as long as it is safe and this is determined by theselectivity of the compound for PPP1R15B.

Myelination in Explants from Neuropathic Mice (FIG. 10). CMT is a groupof myelin neuropathies caused by mutations in a number of genes.Mutations in the peripheral myelin protein PMP22 are the most commoncauses of CMT. A mutation in PMP22 (Trembler-J) causes the misfolding ofPMP22 and a disease in mice that resembles CMT in human due to defectsin myelin in the peripheral nervous system. Explants from PM P22 mutantmice recapitulates the severe hypomyelination observed in the humandiseases. The inventors found that treatment of dorsal root gangliaculture (DRG) from PM P22 mutant mice improved myelination. It has beenpreviously found that the DRG cultures from mutant mice are usefulmodels to predict therapeutic efficacy of compounds. Thus, the datapresent here demonstrate that the compound of Example 1 will be usefulto treat a disease caused by mutation or overproduction of PMP22, suchas CMT disease. The compound of Example 1 will also be useful in thetreatment of other myelin disorders.

Assessment of PPP1R15B Efficacy in a Metabolic Disease (FIG. 11)

It is known that metabolic diseases such as diabetes, obesity, fattyliver disease, and atherosclerosis are associated with pathological ERstress and it is believed that pharmacological modulators of the UPR mayhave therapeutic benefit. As there were no PPP1R15B inhibitors availableprior to this study it was unclear whether PPP1R15B could be atherapeutic target in metabolic diseases. The inventors tested thispossibility and found that treatment of obese mice with a compound ofExample 1 reduced the pathological high blood glucose in these mammals(FIG. 11). This demonstrates that treatment with a compound of Example 1can ameliorate a metabolic disorder. Having shown in one disease modelthat treatment with a compound of Example 1 is beneficial, it is evidentthat the compounds of the invention will be beneficial to othermammalian metabolic disorders such as diabetes, obesity, fatty liverdisease, and atherosclerosis.

Example B

Protein Expression and Purification

MBP-R15A³²⁵⁻⁶³⁶-His and MBP-R15B³⁴⁰⁻⁶⁹⁸-His were expressed and purifiedas described before (Das et al. Science, 2015). cDNA encoding for humanPP1γ was cloned into the baculovirus transfer vector pDW464 to add aN-terminal biotin acceptor peptide (BAP). The vector also encodes forthe E. coli biotin holoenzyme synthetase (BirA), so that BAP-taggedproteins can be biotinylated (bio-PP1c) in vivo in Spodoptere frugiperda(Sf9) insect cells (Duffy et al., Anal. Biochem., 262, 122-128, 1998).The Bac-to-Bac baculovirus expression system (Thermo Fisher Scientific)was used to generate the recombinant bacmid DNA and Sf9 insect cellswere used to amplify the viral stocks. The protein was produced usingSf9 insect cells in Insect-Xpress media (Lonza). bio-PP1c was purifiedby anion exchange chromotography on a HiTrap Q HP column (GEHealthcare), followed by gel filtration (HiLoad 16/600 Superdex 200column, GE Healthcare. The proteins were analyzed on BOLT SDS-PAGE 4-12%Bis-Tris gels (Thermo Fisher Scientific) stained with InstantBlue(Expedeon) and the presence of a biotinylated PP1 was confirmed by awestern blot using a Pierce High Sensitivity Streptavidin-HRP antibody(Thermo Fisher Scientific). This results in a partially pure protein,full purification is reached in later stages due to the high affinityand specificity of the biotin to streptavidin (on the SPR chip).

Surface Plasmon Resonance (SPR)

Capture of Bio-GBZ or Bio-PP1c on the SA Sensor Chip

A Biacore T200 (GE Healthcare) system was used for all experiments andbiotinylated GBZ (bio-GBZ) {Tsaytler:2011ji} or bio-PP1 was captured ona Sensor Chip SA (GE Healthcare). The streptavidin coated surface wasactivated by 1 minute injection with a solution of 50 mM NaOH and 1 MNaCl three times at a flow rate of 10 μl/min. bio-GBZ or bio-PP1c wasdiluted in the running buffer (50 mM Tris pH 7.5, 100 mM NaCl, 0.1 mMEGTA, 0.05% Tween 20, 0.1% DMSO) and injected at approximately 300 nMconcentration at a flow rate of 10 μl/min directly to streptavidincoated surface to reach immobilization level of bio-GBZ or bio-PP1ccorresponding to ˜200 and 6000 RU, respectively. A blank immobilizationwas performed for one of the SA sensor chip surface to use as areference.

Determining Steady-State Binding Constants of Small Molecules to R15Holophosphatase Complexes using the bio-PP1c Surface

Small molecules were stored as 50 mM stock solutions in 100% DMSO. Priorto determining binding constants, serial dilutions of either 12 or 8concentrations of the compounds were prepared in the running buffer in a96-well plate. Prior to each compound dilution series the regulatorysubunit, MBP-R15A³²⁵⁻⁶³⁶-His or MBP-R15B³⁴⁰⁻⁶⁹⁸-His, was diluted to 10μM in the running buffer and captured on the bio-PP1c surface at a flowrate of 30 μl/min for 1 minute to form the holophosphatase complex onthe sensor chip surface. This was followed by 1 minute stabilizationperiod, to wash off any unspecific binding. Then, without regeneratingthe surface, the compound dilution series was injected onto the surfaceof the chip at a flow rate of 30 μl/min for 1 minute, followed by 2minutes dissociation time. After each dilution series the surface wasregenerated using 3 M NaCl for 90 seconds. After regeneration, SPRresponses generally returned to base levels and the bio-PP1c surface wasready for the next compound dilution series. In order to be able tocorrect for small variations in DMSO concentration between samples,eight solvent samples ranging from 0.06 to 8% DMSO were injected every50th cycle. The flow cell temperature was 10° C.

Data Analysis

Sensorgrams were analyzed using the Biacore T200 evaluation software andthe binding constants determined based on a steady-state model. Kineticexperiments are carried out using different concentrations of thecompound and their respective equilibrium binding levels determined.These equilibrium response levels (R_(eq)) are plotted againstconcentration and fitted using a global fit, which is able to determinesteady-state affinity constants, i.e. the concentration at 50%saturation is K_(D). The K_(D) values are shown in Table 1.

Measurement of Cytoprotection Against ER Stress

HeLa cells (40,000 cells/ml) were plated in a 96-well plate and treatedwith different concentrations (0-20 μM) of a compound as indicated inthe presence of 250 ng/ml Tunicamycin for 72 hours. To monitor celldeath 1/2000 dilution of the CelITox green dye (Promega) was added tothe media. The growth of the cells was monitored over time and picturestaken every 2 hours with the IncuCyte ZOOM system and analysed by theIncuCyte ZOOM software (Essen BioScience). Table 1 shows the ability ofthe compounds to protect cells from cytotoxic stress caused bytunicamycin. Cytoprotection against ER stress can be measured by asuitable assay. In this instance, cytoprotection was measured in HeLacells in which ER stress was elicited by the addition of mediacontaining tunicamycin, a drug that blocks N-glycosylation, therebypreventing protein folding and inducing the unfolded protein response.Cell viability was then detected in the presence and absence of acompound listed in Table 1 after a set period of time, by monitoringcell growth over time. Cytoprotection from ER stress was measured interms of the percentage increase in viable cells (relative to control)after ER stress.

${{Growth}\mspace{14mu} {ratio}} = \frac{{Phase}\mspace{14mu} {confluency}\mspace{14mu} (\%)\mspace{14mu} {at}\mspace{14mu} X\mspace{14mu} {hours}}{{Phase}\mspace{14mu} {confluency}\mspace{14mu} (\%)\mspace{14mu} {at}\mspace{14mu} 0\mspace{14mu} {hours}}$${\% \mspace{14mu} {of}\mspace{14mu} {dead}\mspace{14mu} {cells}} = {\frac{{Green}\mspace{14mu} {confluency}\mspace{14mu} (\%)\mspace{14mu} {at}\mspace{14mu} X\mspace{14mu} {hours}}{{Phase}\mspace{14mu} {confluency}\mspace{14mu} (\%)\mspace{14mu} {at}\mspace{14mu} X\mspace{14mu} {hours}} \times 100}$

EC50

EC50 were extrapolated from dose response experiments on cell death.Data from IncuCyte was plotted and analysed in Prism. The “% of celldeath” for each data point. For each concentration there is a % of celldeath vs time, the area under the curve was taken and plotted agains logconc =>EC50 (analysis in Prism).

Assessment of Translation Rates

Cells (90,000 cells/ml) were plated in 12-well plates, treated asindicated, labelled with 100 μCi/mI 35S-methionine (Hartmann Analytic)for 10 minutes at 37° C., washed with ice-cold PBS and lysed in 120 μlLaemmli Buffer. Lysates were boiled at 95° C. for 5 minutes, sonicatedand resolved on 4-12% Bolt Bis-Tris Plus Gels (Thermo FisherScientific). Gels were then stained with InstantBlue (Expedeon) andanalyzed by phosphorimaging and quantified using ImageJ.

By monitoring translation rates as described above, the activities ofR15A, R15B and R15A/B inhibitors can be defined as follows (also shownin FIG. 12). An R15A inhibitor has no effect on translation inunstressed cells but prolongs translation attenuation following stress.A selective R15B inhibitor transiently attenuate protein synthesisbecause R15A compensates for the inhibition of R15B. An R15A/B inhibitorpersistently inhibits protein synthesis.

Translation can be inhibited at many levels. To ensure that the R15A andB inhibitors are on-target, we monitor the levels of ATF4 in cellstreated with compounds. A R15A/B inhibitor persistently induce elF2αphosphorylation and elF2α phosphorylation results in ATF4 translation.We show here that an A/B inhibitor indeed induces ATF4, confirmingtarget engagement in cells.

TABLE 1 Selective binding towards Cytoprotection ⇒ R15A or from Tm maxEffect ⇒ Compound R15B (based (compared to at on cell EC50 Effect on ⇒(E) R15A R15B PP1 on SPR) GNB) [μM] death [μM] translation Inhibits 1 8.32 ± 0.14  7.33 ± 1.79 6.28 R15AB 0% — Yes 4.9 R15A/B 2  46.45 ±18.28 9.222 — R15B 0% — Yes 7.709 R15B 3  4.49 ± 1.47 0.149 10.32 R15AB52% 2.5 Yes 9.166 Persistent R15A/B 4 0.457 ± 0.13 0.022 ± 0.02 331.6R15AB 80% 20 No — Persistent/ R15A/B Transient 5  8.45 ± 0.41  2.67 ±0.40 30.59 R15AB 48% 5 Yes 9.458 R15A/B 6 5.427 ± 1.10 9.094 ± 1.37 —R15AB 61% 2.5 Yes 9.653 Persistent R15A/B 7 10.20 ± 2.57 4.336 ± 2.3512.98 R15AB 46% 5 Yes 11.5 R15AB 8  6.37 ± 1.03 11.34 10.59 ± 3.82 R15AB0% — Yes 11.68 R15AB 9 13.6 ± 0.8 39.6 ± 5.9 14.7 ± 1.4 R15A 67% 5 Yes11.98 Off target 10 0.391 ± 0.14 0.440 ± 0.09 R15AB 85% 0.625 Yes 12.52Persistent R15A/B 11 16.2 ± 2.1 13.38 ± 0.47 R15AB 34% 5 Yes 12.7 R15AB12 7.24 ± 0.5 11.7 ± 0.3 30.7 ± 1.5 R15AB 51% 5 Yes 15.13 PersistentR15A/B 13 4.624 ± 0.59  6.684 ± 2.343 — R15AB 73% 5 Yes 15.48 PersistentR15AB 14 5.446 ± 1.48 5.434 ± 1.26 — R15AB 100% 2.5 Yes 18.4 PersistentR15A/B 15 11.17 7.681 ± 2.18 6.143 ± 0.28 R15AB 46% 10 Yes 19.5 R15A/B16  1.05 ± 0.013 0.035 ± 0.02 — R15B 92% 2.5 Yes 20.76 Transient R15B 1713.0 ± 0.5 18.1 ± 1.8 12.0 ± 0.8 R15AB 66% 20 No — (No) (R15A/ B) 1810.20 ± 1.62 4.850 ± 0.14 10.17 ± 0.14 R15AB 54% 2.5 Yes 23.8 R15A/ B 205.666 ± 0.72 5.173 ± 0.69 — R15AB 88% 5 Yes 37.83 R15AB 21 0.832 0.537(—) R15AB 41% 20 No — Transient R15B 22 7.622 12.35 — R15AB 48% 20 No —R15AB 23 1.424 1.048 ± 0.32 — R15AB 53% 20 No — R15AB 24 18.13 ± 7.5718.67 6.267 R15AB 62% 20 No — R15AB 25  6.18 ± 3.31 9.223 ± 0.19 3.65R15AB 54% 20 No — 26 23.1 ± 0.9 30.0 ± 2.3 R15AB 57% 10 No — R15AB 2710.56 ? — 57% 10 No — R15AB 29 7.873 ± 1.48 4.057 — R15AB 62% 10 No —R15AB 30 5.15 ± 1.7 12.3 ± 0.6  12.8 ± 0.08 R15AB 85% 10 No — No R15A/B31 11.1 ± 1.6 14.3 ± 0.7 12.6 ± 1.4 R15AB 99% 10 No — No R15A 32 4.818 ±0.26 17.28 — 100% 5 No — Persistent R15A/B 34 17.2 ± 1.9 31.3 ± 2.0R15AB 57% 5 No — R15AB GBZ + 16 100% 1.25 Yes 12.6 Persistent R15A/B

1. A compound of formula IA:

or a pharmaceutically acceptable salt thereof, wherein: X^(a) is N orCR^(2a); Y^(a) is N or CR^(4a); R^(1a) is H, F, Cl or Br; R^(2a),R^(3a), R^(4a), R^(5a) each independently represent H, F or Cl; with theproviso that: when X^(a) and Y^(a) represent CH: R^(1a) is not F, Cl orBr when R^(3a) and R^(5a) both represent H; R^(5a) is not F or Cl whenR^(1a) is Cl and R^(2a) is H; R^(3a) is not F when R^(1a) is Cl andR^(5a) is H; R^(3a) is not Cl when R^(1a) and R^(5a) are both H or whenR^(1a) is Cl and R^(5a) is H; R^(1a), R^(3a) and R^(5a) are not all H;or R^(1a) is not Cl when R^(3a) is H and R^(5a) is F; when X^(a)represents CH and Y^(a) represents CR^(4a) wherein R^(4a) is Cl: R^(1a)and R^(5a) are not both Cl; R^(3a) is not Cl when R^(1a) and R^(5a) areCl; when X^(a) represents CR^(2a) and Y^(a) represents CR^(4a) andR^(2a) and R^(4a) are both Cl, R^(1a), R^(3a) and R^(5a) are not all H.2. The compound according to claim 1, wherein X^(a) represents CR^(2a)and Y^(a) represents CR^(4a), wherein R^(2a) and R^(4a) eachindependently represent H, F or Cl.
 3. The compound according to claim1, wherein three of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) representCl or F and two of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) representH; or wherein three of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) arepresent Cl and two of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a)represent H; or wherein R^(3a) is Cl or F, and R^(1a), R^(2a), R^(4a)and R^(5a) are independently selected from H, F and Cl, wherein two ofR^(1a), R^(2a), R^(4a) and R^(5a) are selected from F and Cl and two ofR^(1a), R^(2a), R^(4a) and R^(5a) represent H. 4-5. (canceled)
 6. Thecompound according to claim 1, wherein R^(5a) represents H.
 7. Thecompound according to claim 1, wherein the compound is in the E-isomerform.
 8. The compound according to claim 1, wherein the compound isselected from:(E)-2-((2,4-dichloropyrimidin-5-yl)methylene)hydrazine-1-carboximidamide;(E)-2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(3,5-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,3,4-trichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,4-dichloro-5-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(4-chloro-3-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2-bromo-3-chlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,3,5-trichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(3,4-dichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,4-dichloro-3-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,3-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(3-chloro-4-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,3-dichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(4,5-dichloro-2-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2-chloro-5-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-((2-chloropyridin-3-yl)methylene)hydrazine-1-carboximidamide;(E)-2-((2,6-dichloropyridin-3-yl)methylene)hydrazine-1-carboximidamide;(E)-2-(3-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(3-chlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2-chloro-3-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,5-dichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2-chloro-3,4-difluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2-chloro-3,5-difluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(3-chloro-2-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,3,6-trichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2-chloro-4,5-difluorobenzylidene)hydrazine-1-carboximidamide; or(E)-2-(3,5-dichlorobenzylidene)hydrazine-1-carboximidamide; or apharmaceutically acceptable salt thereof.
 9. A compound of formula IBfor use the treatment of a disease state alleviated by the inhibition ofPPP1R15A and PPP1R15B,

or a pharmaceutically acceptable salt thereof, wherein: X^(b) is N orCR^(2b); Y^(b) is N or CR⁴; R^(1b) is H, F, Cl or Br; R^(2b), R^(3b),R^(4b), R^(5b) each independently represent H, F, or Cl; with theproviso that: when X^(b) and Y^(b) both represent N, R^(1b) and R^(3b)are not both Cl; R^(1b) is not Br when X^(b) is CR^(2b) and R^(2b) isCl; when R^(3b) and R^(4b) are both H, R^(1b) and R^(2b) are not bothCl; when X^(b) is CR^(2b) and R^(2b) and R^(3b) are both H, R^(1b) isnot Cl when Y^(b) is CR^(4b) and R^(4b) is F; when X^(b) is CR^(2b) andY^(b) is CR^(4b), R^(1b), R^(2b), R^(3b),R^(4b) and R^(5b) are not allH; when X^(b) is CR^(2b) and Y^(b) is CR^(4b) and R^(2b), R^(3b), R^(4b)and R^(5b) are H, R^(1b) is not Cl; when X^(b) is CR^(2b) and Y^(b) isCR^(4b) and R^(2b), R^(3b) and R^(4b) are H, R^(1b) is not Cl whenR^(5b) is F; when X^(b) is CR^(2b) wherein R^(2b) is Cl and Y^(b) isCR^(4b) wherein R^(4b) is H, R^(1b) is not Cl when R^(3b), R^(4b) andR^(5b) are H, or when R^(3b) and R^(4b) are H and R^(5b) is Cl.
 10. Thecompound according to claim 9, wherein X^(b) represents CR^(2b) andY^(b) represents CR^(4b), wherein R^(2b) and R^(4b) each independentlyrepresent H, F or Cl.
 11. The compound according to claim 9, whereinthree of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b) represent Cl or F andtwo of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b) represent H; or whereinthree of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b) represent Cl and twoof R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b) represent H; or whereinR^(3b) is Cl or F and R^(1b), R^(2b), R^(4b) and R^(5b) areindependently selected from H, F and Cl, wherein two of R^(1b), R^(2b),R^(4b) and R^(5b) are selected from F and Cl and two of R^(1b), R^(2b),R^(4b) and R^(5b) is H. 12-13. (canceled)
 14. The compound according toclaim 9, wherein R^(5b) is H.
 15. The compound according to claim 9,wherein the compound is in the E-isomer form.
 16. The compound accordingto claim 9, wherein the compound is selected from:(E)-2-((4-chlorophenyl)methylene)hydrazine-1-carboximidamide;(E)-2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(3,5-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,3,4-trichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,4-dichloro-5-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(4-chloro-3-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,3,5-trichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(3,4-dichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,4-dichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,4-dichloro-3-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,3-dichloro-4-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(3-chloro-4-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(4,5-dichloro-2-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-((2-chloropyridin-3-yl)methylene)hydrazine-1-carboximidamide;(E)-2-((2,6-dichloropyridin-3-yl)methylene)hydrazine-1-carboximidamide;(E)-2-(3-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(3-chlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2-chloro-3-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2,5-dichlorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2-chloro-3,4-difluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2-chloro-3,5-difluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(3-chloro-2-fluorobenzylidene)hydrazine-1-carboximidamide;(E)-2-(2-chloro-4,5-difluorobenzylidene)hydrazine-1-carboximidamide; ora pharmaceutically acceptable salt thereof.
 17. The compound accordingto claim 1, wherein the compound is(E)-2-(3,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide or apharmaceutically acceptable salt thereof.
 18. The compound according toclaim 1, wherein the compound is(E)-2-(2,4,5-trichlorobenzylidene)hydrazine-1-carboximidamide or apharmaceutically acceptable salt thereof.
 19. A method of treating adisorder of a subject, the method comprising administering atherapeutically effective amount of the compound according to claim 1 tothe subject, wherein the disorder is a disorder associated withaccumulation of misfolded proteins or a proteostatsis disorder.
 20. Themethod according to claim 19, wherein the disorder is associated withPPP1R15A and PPP1R15B.
 21. A method of treating a disorder of a subject,the method comprising administering a therapeutically effective amountof the compound according to, claim 9 to the subject, wherein thedisorder is a disorder associated with accumulation of misfoldedproteins or a proteostatsis disorder.
 22. The method according to claim19, wherein the disorder is selected from Alzheimer's disease,Parkinson's disease, Huntington's disease, or another otherpolyglutamine disorder or tauopathy.
 23. A pharmaceutical compositioncomprising the compound of claim 1 and a pharmaceutically acceptableexcipient. 24-25. (canceled)
 26. The compound according to claim 1,wherein three of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represent Clor F and two of R^(1a), R^(2a), R^(3a), R^(4a) and R^(5a) represent H,and wherein at least one of R^(1a), R^(2a), R^(3a), R^(4a)and R^(5a) isF.
 27. The compound according to claim 9, wherein three of R^(1b),R^(2b), R^(3b), R^(4b) and R^(5b) represent Cl or F and two of R^(1b),R^(2b), _(R) 3b , R^(3b) and R^(5b) represent H, and wherein at leastone of R^(1b), R^(2b), R^(3b), R^(4b) and R^(5b) represents F.